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

1. Basics of Split-and-pool Synthesis
In order to efficiently generate a large number of compounds on solid phase, split-and-pool synthesis is often employed as a general strategy. As shown in Figure 4, tentagel beads are first&#160;split into three portions. Each portion is reacted with a different reagent, generating the first residue on beads. After the first reaction, all three portions are pooled together, mixed, and then split again into three portions. Each portion will again react with a different reagent, generating the second residue on beads. After two split-and-pool steps, nine compounds are generated.
In sub-monomer synthesis, beads are first divided into several portions to react with different bromo acids in the presence of coupling reagent. After washing with solvent, all beads will be pooled together and mixed, then again divided into several portions to react with different primary amines. After amination, all beads are pooled together and washed thoroughly, completing a full monomer on each bead. This process can be repeated till desired diversity is reached.
2. Preparation of Acid Bromide from Natural Amino Acids
In sub-monomer synthesis, the synthesis of each monomer is divided into two separate steps: 1. coupling of acid bromide and 2. amination with primary amines (Figure 2). In order to synthesize a peptide tertiary amide, chiral acid bromides with side chains on the alpha carbon will be prepared from natural amino acids. Here we describe the method of transforming a natural amino acid into the corresponding acid bromide with high stereo fidelity. We use alanine as an example; other amino acids including serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, valine, isoleucine, phenylalanine can also be transformed into bromo acids under similar conditions. Note that some of the amino acids with functional groups like phenol, guanidine and amine need to be protected before the transformation. The reaction setup is shown in Figure 3.
Safety Precaution: For the following reactions involving HBr, NaNO2 and other corrosive/toxic chemicals, proper safety equipment like safety goggles, lab coat, and chemical resistant gloves are needed. All reactions should be performed in a fume hood by experienced chemist.
<ol>
	<li>Add 370 ml water into 630 ml 48% HBr solution to prepare a 1 L, 30% HBr solution. Add 500 ml ethylene glycol into a 1 L bath container; add dry ice to maintain the temperature at -10 &#176;C. Caution: 48% HBr solution is strongly acidic and corrosive, handle with care. Read the MSDS before use.</li>
	<li>Add D-alanine (8.9 g, 0.1 mol) and KBr (11.9 g, 0.1 mol) to a 250 ml three-neck round bottom flask with a magnetic stir bar. Add 100 ml, and the 30% HBr prepared in the previous step. Put the flask in the ethylene glycol bath prepared in step 2.1 and keep the temperature at -10 &#176;C. Bubble argon through a long needle from the bottom of the flask for 10 min as shown in Figure 3. Stir the solution with the magnetic stir bar at 300 rpm.</li>
	<li>Dissolve NaNO2 (8.28 g, 0.12 mol) in a 100 ml beaker with 20 ml water. Add the solution into the pressure equalizing dropping funnel and seal the dropping funnel with a septum. Slowly turn on the valve of the dropping funnel and let the NaNO2 solution drop into the flask. Control the valve to adjust the dripping rate to approximately 2 drops per sec. Keep the stirring at 300 rpm and keep the argon bubbling&#160;from the bottom of the flask. The flask should be kept in ethylene glycol bath at -10 &#176;C until all NaNO2 is added. Caution: This step generates heat and gas during the addition of NaNO2 solution. Dripping rate should be carefully controlled and the whole system should be open through the argon outlet.</li>
	<li>Keep stirring for 3 more hr&#160;and let the temperature warm up from -10 &#176;C to room temperature. The resulting solution should be clear to light yellow;&#160;if the color is too dark, apply vacuum to remove the excess nitrogen oxides and possible Br2 generated during the reaction.</li>
	<li>Extract the product from the solution with 3x 35 ml&#160;diethyl ether&#160;using an extracting funnel. Combine the organic phase and wash it with saturated brine. The organic phase can also be washed with a small amount of NaHCO3 prior to washing with brine to remove the color if it is dark. Dry the organic phase over Na2SO4 for 6 hr.</li>
	<li>Filter out the Na2SO4 and evaporate the solvent under vacuum, crude product should be obtained as clear to pale yellow oil. Crude product can be further purified by distillation at 115 &#176;C, 3 mm Hg, or by silica column with 3:1 hexane:ethyl acetate.</li>
	<li>Pure product is obtained as clear oil, 6.6 g (yield 74%), density = 1.69 g/ml, [&#945;]D20 = +24&#176; (methanol), 1H NMR (400 MHz, CDCl3) &#948; 4.41 (q, J = 7.0 Hz, 1H), 1.86 (d, J = 7.0 Hz, 3H). In the case of (S)-2-bromopropanoic acid-d4 (prepared from d4-L-alanine), pure product is obtained as clear oil, yield 78%, density = 1.72 g/ml, [&#945;]D20&#160;= -19&#176; (methanol). 1H NMR, no significant H signal is observed. ESI-MS- [M-1]-&#160;= 155.1 (expected 154.97). For (S)-2-bromo-4-methylpentanoic acid (prepared from L-leucine using the same procedure) pure product is obtained as clear oil, yield 89%, [&#945;]D20&#160;= +37&#176; (methanol), 1H NMR (400 MHz, CDCl3) &#948; 4.30 (t, J = 7.7 Hz, 1H), 1.94 (dd, J = 10.8, 3.9 Hz, 2H), 1.81 (tt, J = 13.2, 6.5 Hz, 1H), 0.96 (dd, J = 18.2, 6.6 Hz, 7H). In the case of (S)-2-bromo-3-phenylpropanoic acid (prepared from L-phenylalanine using the same procedure) pure product is obtained as pale yellow oil, yield 72%, [&#945;]D20&#160;= +17&#176; (methanol), 1H NMR (400 MHz, CDCl3) &#948; 7.38 &#8211; 7.19 (m, 5H), 4.42 (dd, J = 8.1, 7.3 Hz, 1H), 3.47 (dd, J = 14.2, 8.2 Hz, 1H), 3.25 (dd, J = 14.2, 7.2 Hz, 1H).</li>
</ol>
3. Isotopic Labeling of Alanine Using Transaminase
In combinatorial library synthesis, especially in the split-and-pool synthesis of one-bead one-compound (OBOC) libraries, the amount of compound that can be obtained from each bead is relatively small. (Typically 1 pmol to 10 nmol). Additionally, mass spectrometry is widely used for the identification and characterization of the final compound due to its high sensitivity. In order to use mass spectrometry to determine the absolute stereochemistry at the chiral centers of the final PTA products, bromo acid enantiomers should be isotopically labeled before use. Here we describe the method of using transaminase and D2O to label L-alanine.
<ol>
	<li>Dissolve L-alanine (300 mg, 3.36 mmol) with 10 ml of D2O in a 50 ml polyethylene tube. Add &#945;-ketoglutarate (10 mg, 0.068 mmol) as co-substrate. Warm up the tube to 37 &#176;C and adjust the pD to 8.5-8.7 using a 1 M NaOD soution. Note: pD is determined by pH test strips. Traditional electro pH meter equipped with glass electrode selective for H+ may give incorrect read out for D+.</li>
	<li>Add alanine transaminase (0.1 mg, EC 2.6.1.2 from pig heart, Roche Diagnostics, Indianapolis, IN) to the pD 8.5 - 8.7, 37 &#176;C solution prepared from previous step. Put the tube in a 37 &#176;C incubator and incubate it overnight with mild shaking, 10 to 30 rpm is preferred.</li>
	<li>After overnight incubation, take 0.5 ml of the D2O solution and check the reaction progress by 1H-NMR. All proton signals of alanine, &#948; 3.76 (q, J = 7.2 Hz, 1H), 1.46 (d, J = 7.3 Hz, 3H), 1H NMR 400 MHz, should be greatly suppressed due to deuteration. More than 98% of the proton should be exchanged to deuterium as described previously23. Note: D2O can be partially recovered by distillation if the reaction is performed on large scale (&#62;200 ml D2O). Normally, 60% to 80% of D2O can be distilled from the solution.</li>
	<li>Freeze the above solution with liquid nitrogen and lyophilize it using a lyophilizer to obtain white deuterated L-alanine powder.</li>
</ol>
4. Synthesis of Peptoid Linker Region
The linker region is not required for PTA library synthesis. However, in order to avoid the high background in the lower molecular weight range (100-600) of MALDI mass spectroscopy and to improve the ionization of the compounds, a peptoid linker with multiple polar residues is often used. This peptoid linker can be synthesized through standard peptoid synthesis procedure. Here we will synthesize a pentamer of N-methoxyethyl glycine as the linker (as shown in Figure 5).
<ol>
	<li>Swell 90 &#956;m Tentagel beads with RAM linker (1 g, 0.27 mmol/g) in 10 ml DMF for 3 hr&#160;in a 12 ml syringe reactor with mild shaking.</li>
	<li>Drain the DMF from the reactor and add 10 ml 20% piperidine DMF solution to deprotect the fmoc group from the Rink amide linker. Shake the beads with 20% piperidine solution for 30 min. Wash with DMF 5x&#160;to remove all piperidine.</li>
	<li>Take a few beads out from the syringe and test it with chloranil test. Beads should turn dark brown (chloranil test positive for primary amine) if fmoc is successfully deprotected.</li>
	<li>Prepare the following solutions: 1. 20 ml, 2 M bromoacetic acid/DMF solution; 2. 20 ml, 2 M DIC/DMF solution; 3. 10 ml, 1 M methoxylethylamine/DMF solution.</li>
	<li>Add 5 ml of 2 M bromoacetic acid/DMF solution to the beads, shake gently. Then add 5 ml of 2 M DIC/DMF solution to the beads; seal the syringe with the plunger and put it on the shaker. Shake for 10 min.</li>
	<li>Wash the beads with DMF thoroughly. Add 2 ml of 1 M methoxyethylamine/DMF solution prepared from step 4.4 to the beads. Seal the syringe with the plunger and shake it on the shaker for 30 min.</li>
	<li>Wash the beads with DMF 5x. Check a few beads with chloranil test, if positive (beads turn blue), then continue to the next step. Otherwise, repeat step 4.6.</li>
	<li>Repeat steps 4.5 to 4.7, 4x&#160;to complete the pentamer.</li>
</ol>
5. Split-and-pool Synthesis of PTA Library with (R)- and (S)-2-bromopropionic Acids
Here we describe the synthesis of a small PTA library with a theoretical diversity of 9,261 compounds using the 1 g&#160;of beads from step 4.8. Note that a 90 &#181;m tentagel bead contains approximately 2.9 million beads per gram; therefore the redundancy of the library will be 2.9 x 106 / 9,261 = 312 copies. We will use bromoacetic acid, (R)-2-bromopropanoic and isotopic labeled (S)-2-bromopropanoic acid-d4 as the acids, and 7 different amines (A1 ~ A7, see Figure 5 for details) for amination. Syringe reactors and a vacuum manifold will be used to perform the synthesis.
<ol>
	<li>Add 10 ml of 1:1 DCM:DMF to the syringe from step 4.8; use a 1,000 &#956;l pipette with a truncated pipette tip to split all 1 g&#160;beads evenly into three 5 ml syringe reactors. Label them as B (bromoacetic acid), R ((R)-2-bromopropanoic) and S ((S)-2-bromopropanoic acid-d4). Wash all 3 syringes with DCM 3x, and wash syringes labeled with R and S with anhydrous THF 3x, wash syringe labeled with B with DMF 3x.</li>
	<li>Syringe R and S. BTC coupling of bromopropanoic acid.
	<ol>
		<li>Prepare a fresh BTC/THF solution. Add approximately 200 mg of BTC into the vial in a fume hood, seal it with the cap. Weigh the amount of BTC in the vial. Calculate the amount of solvent needed and add anhydrous THF into the vial to make a 20 mg/ml BTC/THF solution.</li>
		<li>Prepare the bromo acids/BTC mixture. Add (R)-2-bromopropanoic acid (89 &#956;l, 0.95 mmol) and (S)-2-bromopropanoic acid-d4 (89 &#956;l, 0.95 mmol) in two small vials separately. To each vial, add 5 ml of the above 20 mg/ml BTC/THF solution. Seal the two vials and put them in -20 &#176;C freezer for 20 min.</li>
		<li>Add 1,125 &#956;l, 2:1 THF/DIPEA (750 &#956;l THF, 375 &#956;l DIPEA, 2.2 mmol) to syringe R and S separately. Mix the beads with the pipette tip. Let them sit for 5 min.</li>
		<li>Take the two cooled bromo acids/BTC mixtures from step 5.2.2, add 2,4,6-Trimethylpyridine (356 &#956;l, 2.7 mmol) to each vials. White precipitates will form immediately. Apply the corresponding suspension directly to the basified beads (syringe R and S in step 5.2.3) as soon as possible and then put them on a shaker to shake under 120 rpm for 2 hr. 
		Note: The solution in the syringe reactors should be a pale yellowish suspension during the whole course of the reaction. A darker color is an indication of excessive heat released during the initial addition of the acid chloride solution. This can be solved by further cooling down or dilute the bromo acids/BTC mixture.</li>
	</ol>
	</li>
	<li>Syringe B. Bromoacetic acid coupling with DIC
	<ol>
		<li>Prepare a fresh 20 ml, 2 M bromoacetic acid/DMF solution. Prepare a 20 ml, 2 M DIC/DMF solution.</li>
		<li>Add 2&#160;ml of 2 M bromoacetic acid/DMF solution to syringe B, shake gently. Add 2&#160;ml of 2 M DIC/DMF solution to syringe B, shake gently.</li>
		<li>Put syringe B on the same shaker as syringe R and S, shake it for 2 hr. Note that the coupling reaction of bromoacetic acid /DIC is done within 30 min; prolonged reaction time is for the convenience of split-and-pool synthesis.</li>
	</ol>
	</li>
	<li>After 2 hr, take syringe R, S and B from the shaker. Wash all three syringes thoroughly with DCM&#160;5x. Then wash with DMF 5x. Note that syringe R and S cannot be washed with DMF before being washed with DCM or THF first.</li>
	<li>Pool all the beads from syringes R, S and B into one 12 ml syringe reactor. Wash all the beads with DMF 5x.</li>
	<li>Add 10 ml of 1:1 DCM:DMF to the syringe; use a 1,000 &#956;l pipette with a truncated pipette tip to split all the beads evenly into 7 individual 2 ml syringes, label them as A1-A7.</li>
	<li>Amination. Prepare 10 ml, 2 M primary amine/DMF solutions for each of the 7 amines listed in Figure 5. Add 5 ml of each amine solution to the corresponding syringe A1-A7. Incubate all 7 syringes in a 60 &#176;C incubator with shaking overnight.</li>
	<li>After incubation, wash all beads thoroughly with DMF.&#160; Take a few of beads from each syringe and check with chloranil test. If the beads turn green (positive) within 3 min, proceed with next step. If negative, repeat step 5.7 for the negative syringes.</li>
	<li>Repeat steps 5.1 to 5.8 2x to complete the trimer. Optional step: After each cycle, we recommend to check the synthesis quality by mass spectroscopy as described below. All 9,261 compounds are now synthesized on tentagel beads as OBOC library.</li>
	<li>Mass spectroscopic confirmation of PTAs. 
	PTAs are highly structured oligomers and possess many common features of N-methylated peptides. One of the common problems of solid phase synthesis of N-methylated peptides is the acid degradation during TFA cleavage. To suppress acid degradation, cleavage of molecules like cyclosporine from solid support is often carried out under low temperature. We compared various cleaving conditions for cleaving different PTA molecules from the solid support. We found that, generally, low temperature and reduced TFA concentration could effectively suppress acid degradation and provide purer compounds.
	<ol>
		<li>Prepare 10 ml 1:1 TFA/DCM solution in a 15 ml tube. Seal the tube and put it in a -20 &#176;C freezer for 20 min.</li>
		<li>Wash the beads that need to be cleaved with DCM 5x. Shake the beads in DCM for 15 min and wash the beads again with DCM 5x.</li>
		<li>Drain the DCM from the syringe. Use a light microscope and a pipette with truncated tip to transfer each individual bead into a 96-well plate, one bead per well.</li>
		<li>Cover the 96-well plate with a cover slip. Put the plate in a -20 &#176;C freezer for 15 min.</li>
		<li>Take the cooled 1:1 TFA/DCM solution from step 5.10.1 and add 20 &#956;l to each of the wells that contains a bead. Put the cover slip back and put the 96-well plate on a shaker in the -20 &#176;C fridge.</li>
		<li>Shake for 20 min. Take the 96-well plate out and peel off the cover slip. Blow-dry the TFA/DCM from each well by blowing air or argon over it. If more than 10 beads are cleaved, a speedvac can be used to dry the TFA/DCM from the whole plate. Note that at this point, not all of the compounds are cleaved off the beads, but the cleaved compounds should be more than enough to perform the mass spectroscopy analysis.</li>
		<li>Add 20 &#956;l 6:4 ACN:H2O solution to dissolve the cleaved compounds from each well. Spot 0.6 &#956;l of each compound solution together with 0.6 &#956;l CHCA MALDI matrix on the MALDI plate.</li>
		<li>Use MALDI mass spectrometry to determine the molecular weight and sequence (MS/MS) of each compound.</li>
	</ol>
	</li>
</ol>
6. Chloranil Test
<ol>
	<li>Prepare the following reagents fresh for each test. Solution A: 2% Chloranil (CAS: 118-75-2) in DMF. Solution B: 2% acetaldehyde (CAS: 75-07-0) in DMF.</li>
	<li>Mix 100 &#956;l of solution A with 100 &#956;l of solution B before the test in a 1.5 ml tube; drop the beads in and gently shake. If the beads turn blue within 5 min, it indicates the presence of secondary amine on the surface of the beads. Primary amines give a dark brown color instead of turning blue.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptoid+ribbon+secondary+structure.">A peptoid ribbon secondary structure.</a> Angewandte Chemie. 52, 5079-5084 (2013).</li><li>Gorske, B. C., Stringer, J. R., Bastian, B. L., Fowler, S. A., Blackwell, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+for+the+design+of+folded+peptoids+revealed+by+a+survey+of+noncovalent+interactions+in+model+systems.">New strategies for the design of folded peptoids revealed by a survey of noncovalent interactions in model systems.</a> J Am Chem Soc. 131, 16555-16567 (2009).</li><li>Stringer, J. R., Crapster, J. A., Guzei, I. A., Blackwell, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraordinarily+robust+polyproline+type+I+peptoid+helices+generated+via+the+incorporation+of+alpha-chiral+aromatic+N-1-naphthylethyl+side+chains.">Extraordinarily robust polyproline type I peptoid helices generated via the incorporation of alpha-chiral aromatic N-1-naphthylethyl side chains.</a> J Am Chem Soc. 133, 15559-15567 (2011).</li><li>Huang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+threaded+loop+conformation+adopted+by+a+family+of+peptoid+nonamers.">A threaded loop conformation adopted by a family of peptoid nonamers.</a> Journal of the American Chemical Society. 128, 1733-1738 (2006).</li><li>Lee, J. H., Kim, H. S., Lim, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Facile+Solid-Phase+Synthesis+of+Conformationally+Constrained+Bicyclic+Peptoids.">Design and Facile Solid-Phase Synthesis of Conformationally Constrained Bicyclic Peptoids.</a> Organic Letters. 13, 5012-5015 (2011).</li><li>Gao, Y., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Screening+of+Stereochemically+Diverse+Combinatorial+Libraries+of+Peptide+Tertiary+Amides.">Synthesis and Screening of Stereochemically Diverse Combinatorial Libraries of Peptide Tertiary Amides.</a> Chem Biol. 20, 360-369 (2013).</li><li>Urban, J., Vaisar, T., Shen, R., Lee, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lability+of+N-alkylated+peptides+towards+TFA+cleavage.">Lability of N-alkylated peptides towards TFA cleavage.</a> Int J Pept Protein Res. 47, 182-189 (1996).</li><li>Rzuczek, S. G., Gao, Y., Tang, Z., Thornton, C. A., Kodadek, T., Disney, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+Modularly+Assembled+Compounds+That+Impart+Bioactivity+Against+an+RNA+Target.">Features of Modularly Assembled Compounds That Impart Bioactivity Against an RNA Target.</a> ACS Chemical Biology. 8 (10), 2312-2321 (2013).</li><li>Thern, B., Rudolph, J., Jung, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triphosgene+as+highly+efficient+reagent+for+the+solid-phase+coupling+of+N-alkylated+amino+acids&#8212;total+synthesis+of+cyclosporin+O.">Triphosgene as highly efficient reagent for the solid-phase coupling of N-alkylated amino acids&#8212;total synthesis of cyclosporin O.</a> Tetrahedron Letters. 43, 5013-5016 (2002).</li><li>Sleebs, M. M., Scanlon, D., Karas, J., Maharani, R., Hughes, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+Synthesis+of+the+Antifungal+Depsipeptide+Petriellin+A.">Total Synthesis of the Antifungal Depsipeptide Petriellin A.</a> J Org Chem. 76, 6686-6693 (2011).</li><li>Vaisar, T., Urban, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-phase+fragmentation+of+protonated+mono-N-methylated+peptides.+Analogy+with+solution-phase+acid-catalyzed+hydrolysis.">Gas-phase fragmentation of protonated mono-N-methylated peptides. Analogy with solution-phase acid-catalyzed hydrolysis.</a> Journal of Mass Spectrometry. 33, 505-524 (1998).</li><li>Creighton, C. J., Romoff, T. T., Bu, J. H., Goodman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+studies+of+an+unusual+amide+bond+scission.">Mechanistic studies of an unusual amide bond scission.</a> Journal of the American Chemical Society. 121, 6786-6791 (1999).</li><li>Sewald, N., Sewald, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient,+racemization-free+peptide+coupling+of+N-alkyl+amino+acids+by+using+amino+acid+chlorides+generated+in+situ--total+syntheses+of+the+cyclopeptides+cyclosporin+O+and+omphalotin+A.">Efficient, racemization-free peptide coupling of N-alkyl amino acids by using amino acid chlorides generated in situ--total syntheses of the cyclopeptides cyclosporin O and omphalotin A.</a> Angewandte Chemie (International ed. in English). 41, 4661-4663 (2002).</li><li>Astle, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seamless+Bead+to+Microarray+Screening:+Rapid+Identification+of+the+Highest+Affinity+Protein+Ligands+from+Large+Combinatorial+Libraries.">Seamless Bead to Microarray Screening: Rapid Identification of the Highest Affinity Protein Ligands from Large Combinatorial Libraries.</a> Chem Biol. 17, 38-45 (2010).</li><li>Strohalm, M., Kavan, D., Novak, P., Volny, M., Havlicek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mMass+3:+a+cross-platform+software+environment+for+precise+analysis+of+mass+spectrometric+data.">mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data.</a> Anal Chem. 82, 4648-4651 (2010).</li></ol>

The authors declare that they have no competing financial interests.

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

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

<table><tbody><tr><td>2,4,6 trimethylpyridine</td><td>ACROS</td><td>161950010</td><td>CAS:108-75-8</td></tr><tr><td>2-morpholinoethanamine</td><td>Sigma-Aldrich</td><td>06680</td><td>CAS:2038-03-1&amp;nbsp;&amp;nbsp;</td></tr><tr><td>48% HBr water solution</td><td>ALFA AESAR</td><td>AA14036AT</td><td>CAS:10035-10-6</td></tr><tr><td>Acetaldehyde</td><td>Sigma-Aldrich</td><td>402788</td><td>CAS:75-07-0&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>SR015AA-19PS</td><td>CAS:75-05-8</td></tr><tr><td>Anhydrous tetrahydrofuran (THF)</td><td>EMD</td><td>EM-TX0277-6</td><td>CAS:109-99-9</td></tr><tr><td>Benzylamine</td><td>Sigma-Aldrich</td><td>185701</td><td>CAS:100-46-9</td></tr><tr><td>bis(Trichloromethyl) carbonate (BTC)</td><td>ACROS</td><td>258950050</td><td>CAS:32315-10-9</td></tr><tr><td>Bromoacetic acid</td><td>ACROS</td><td>106570010</td><td>CAS:79-08-3</td></tr><tr><td>Chloranil</td><td>Sigma-Aldrich</td><td>23290</td><td>CAS:118-75-2</td></tr><tr><td>Cyclohexanemethylamine</td><td>Sigma-Aldrich</td><td>101842</td><td>CAS:3218-02-8</td></tr><tr><td>D&lt;sub&gt;2&lt;/sub&gt;O</td><td>Cambridge Isotope</td><td>DLM-4-99.8-1000</td><td>CAS:7789-20-0</td></tr><tr><td>D-Alanine</td><td>Anaspec</td><td>61387-100</td><td>CAS:338-69-2</td></tr><tr><td>Dichloromethane (DCM)</td><td>Fisher</td><td>BJ-NS300-20</td><td>CAS:75-09-2</td></tr><tr><td>Dimethylformamide (DMF)</td><td>Fisher</td><td>BJ-076-4</td><td>CAS:68-12-2</td></tr><tr><td>Ethylene glycol</td><td>Oakwood</td><td>44710</td><td>CAS:107-21-1</td></tr><tr><td>Isopentylamine</td><td>Sigma-Aldrich</td><td>W321907</td><td>CAS:107-85-7</td></tr><tr><td>KBr</td><td>ACROS</td><td>424070025</td><td>CAS:7758-02-3</td></tr><tr><td>L-Alanine</td><td>Anaspec</td><td>61385-100</td><td>CAS:56-41-7</td></tr><tr><td>3-Methoxypropylamine</td><td>Sigma-Aldrich</td><td>M25007</td><td>CAS:5332-73-0</td></tr><tr><td>2-Methoxyethylamine</td><td>Sigma-Aldrich</td><td>143693</td><td>CAS:109-85-3</td></tr><tr><td>N-(3-Aminopropyl)-2-pyrrolidinone</td><td>Sigma-Aldrich</td><td>136565</td><td>CAS:7663-77-6&amp;nbsp;</td></tr><tr><td>N,N&#039;-Diisopropylcarbodiimide (DIC)</td><td>ACROS</td><td>115211000</td><td>CAS:693-13-0</td></tr><tr><td>N,N-Diisopropylethylamine (DIPEA)</td><td>Sigma-Aldrich</td><td>D125806</td><td>CAS:7087-68-5</td></tr><tr><td>NaNO&lt;sub&gt;2&lt;/sub&gt;</td><td>ACROS</td><td>424340010</td><td>CAS:7631-99-4</td></tr><tr><td>NAOD 40% solution in water</td><td>ACROS</td><td>200058-506</td><td>CAS:7732-18-5</td></tr><tr><td>Piperidine</td><td>ALFA AESAR</td><td>A12442-AE</td><td>CAS:110-89-4</td></tr><tr><td>Piperonylamine</td><td>Sigma-Aldrich</td><td>P49503</td><td>CAS:2620-50-0</td></tr><tr><td>Propylamine</td><td>Sigma-Aldrich</td><td>240958</td><td>CAS:107-10-8</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>299537</td><td>CAS:76-05-1</td></tr><tr><td>&amp;alpha;-Cyano-4-hydroxycinnamic acid</td><td>Sigma-Aldrich</td><td>39468</td><td>CAS:28166-41-8&amp;nbsp;&amp;nbsp;</td></tr><tr><td>&amp;alpha;-Ketoglutarate</td><td>ALFA AESAR</td><td>AAA10256-22</td><td>CAS:328-50-7</td></tr><tr><td>Tentagel Resin with RINK linker</td><td>Rapp-Polymere</td><td>S30023</td><td></td></tr><tr><td>Alanine transaminase</td><td>Roche</td><td>10105589001</td><td>AKA: Glutamate-Pyruvate Transaminase (GPT)</td></tr><tr><td>Incubator</td><td>New Brunswick Scientific</td><td>Innova44</td><td></td></tr><tr><td>NMR</td><td>Bruker</td><td>400 MHz</td><td></td></tr><tr><td>MALDI mass spectrometer</td><td>Applied Biosystems</td><td>4800 MALDI-TOF/TOF</td><td></td></tr><tr><td>Lyophilizer</td><td>SP Scientific</td><td>VirTis benchtop K</td><td></td></tr><tr><td>Syringe reactor</td><td>INTAVIS</td><td>Reaction Column</td><td>3 ml, 5 ml, 10 ml, 20 ml</td></tr><tr><td>Vacuum manifold</td><td>Promega</td><td>A7231</td><td>Vac-Man</td></tr></tbody></table>

split-and-pool synthesis and characterization of peptide tertiary amide library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

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

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

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

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18.0K Views.  The Scripps Research Institute. Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

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

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 ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

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 and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

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 ...

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 ...