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

Summary

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.

Abstract

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 α-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.

Introduction

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 proteolysis^1-3. Due to the oligomeric nature of these compounds, large synthetic libraries can be readily accessed through monomeric or sub-monomeric synthetic routes^4-7. One of the most studied classes of peptidomimetics is peptoids. Peptoids are oligomers of N-alkylated glycines that can be synthesized easily using a sub-monomer strategy^8,9. Many useful protein ligands ....

Protocol

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 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.

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,4,6 trimethylpyridine	ACROS	161950010	CAS:108-75-8
2-morpholinoethanamine	Sigma-Aldrich	06680	CAS:2038-03-1
48% HBr water solution	ALFA AESAR	AA14036AT	CAS:10035-10-6
Acetaldehyde	Sigma-Aldrich	402788	CAS:75-07-0
Acetonitrile	Fisher	SR015AA-19PS	CAS:75-05-8
Anhydrous tetrahydrofuran (THF)	EMD	EM-TX0277-6	CAS:109-99-9
Benzylamine	Sigma-Aldrich	185701	CAS:100-46-9
bis(Trichloromethyl) carbonate (BTC)	ACROS	258950050	CAS:32315-10-9
Bromoacetic acid	ACROS	106570010	CAS:79-08-3
Chloranil	Sigma-Aldrich	23290	CAS:118-75-2
Cyclohexanemethylamine	Sigma-Aldrich	101842	CAS:3218-02-8
D2O	Cambridge Isotope	DLM-4-99.8-1000	CAS:7789-20-0
D-Alanine	Anaspec	61387-100	CAS:338-69-2
Dichloromethane (DCM)	Fisher	BJ-NS300-20	CAS:75-09-2
Dimethylformamide (DMF)	Fisher	BJ-076-4	CAS:68-12-2
Ethylene glycol	Oakwood	44710	CAS:107-21-1
Isopentylamine	Sigma-Aldrich	W321907	CAS:107-85-7
KBr	ACROS	424070025	CAS:7758-02-3
L-Alanine	Anaspec	61385-100	CAS:56-41-7
3-Methoxypropylamine	Sigma-Aldrich	M25007	CAS:5332-73-0
2-Methoxyethylamine	Sigma-Aldrich	143693	CAS:109-85-3
N-(3-Aminopropyl)-2-pyrrolidinone	Sigma-Aldrich	136565	CAS:7663-77-6
N,N'-Diisopropylcarbodiimide (DIC)	ACROS	115211000	CAS:693-13-0
N,N-Diisopropylethylamine (DIPEA)	Sigma-Aldrich	D125806	CAS:7087-68-5
NaNO2	ACROS	424340010	CAS:7631-99-4
NAOD 40% solution in water	ACROS	200058-506	CAS:7732-18-5
Piperidine	ALFA AESAR	A12442-AE	CAS:110-89-4
Piperonylamine	Sigma-Aldrich	P49503	CAS:2620-50-0
Propylamine	Sigma-Aldrich	240958	CAS:107-10-8
Trifluoroacetic acid	Sigma-Aldrich	299537	CAS:76-05-1
α-Cyano-4-hydroxycinnamic acid	Sigma-Aldrich	39468	CAS:28166-41-8
α-Ketoglutarate	ALFA AESAR	AAA10256-22	CAS:328-50-7
Tentagel Resin with RINK linker	Rapp-Polymere	S30023
Alanine transaminase	Roche	10105589001	AKA: Glutamate-Pyruvate Transaminase (GPT)
Incubator	New Brunswick Scientific	Innova44
NMR	Bruker	400 MHz
MALDI mass spectrometer	Applied Biosystems	4800 MALDI-TOF/TOF
Lyophilizer	SP Scientific	VirTis benchtop K
Syringe reactor	INTAVIS	Reaction Column	3 ml, 5 ml, 10 ml, 20 ml
Vacuum manifold	Promega	A7231	Vac-Man