Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Summary

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

Abstract

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.

Introduction

Peptoids are a class of sequence-controlled polymers with backbone structures mimicking the structure of peptides. Peptoids can be synthesized from diverse amines, which enables peptoids to exhibit highly tunable properties^1,2. Peptoids have been used as molecular models for biophysical research, considered as therapeutic agents, and designed as ligands for proteins^3,4,5,6. Peptoids have been developed into a variety of biologically active compounds, such as anti-fouling and antibody....

Protocol

1. Synthesis of Peptoid

NOTE: The synthesis begins with activating the resin by swelling the resin and removing the protecting group. This is followed by growing the peptoid chain onto the resin through repeating monomer addition cycles. The first monomer coupled to the resin is the C-terminal residue. The peptoid is elongated from the C-terminus to the N-terminus. Once the desired peptoid sequence is achieved, the resin is cleaved off and the peptoid product is purified.

1. Preparation of reagents
   NOTE: The liquid reagents are measured using a micropipette and the solid reagents are measured using an analyt....

Results

The structure of a 9-mer peptoid with N-terminal acetylation, Ac-Nae-(Npe-Nme)₄-NH₂, is shown in Figure 1. The peptoid was synthesized manually in a fritted polypropylene reaction vessel via solid phase approach. Rink amide resin (0.047 mmol, 84 mg with loading 0.56 mmol/g) is used as the solid support to yield the peptoid with an amidated C-terminus. The peptoid chain is built by multiple cycles of monomer addition. Each monomer additio.......

Discussion

A nonamer peptoid, Ac-Nae-(Npe-Nme)₄-NH₂, has been synthesized using the protocol presented. The synthesis apparatus involves a syringe-like polypropylene solid-phase reaction vessel and a mechanical shaker. The reaction vessels are commercially available and low cost. A mechanical shaker is a common apparatus in chemistry laboratories. With the use of a syringe-like reaction vessel, solutions can be drawn into and pushed out of the vessel by manually moving the plunger. This technique allows the mo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ESI-triple quadrupole mass spectrometer, Varian 320L	Agilent Technologies Inc.		The mass spectrometer was acquired from Varian, Inc.
Varian MS workstation, Version 6.9.2, a data acquisition and data review software	Varian Inc.		The software is a part of the Varian 320L package
Burrell Scientific Wrist-action shaker, Model 75 DD	Fisher Scientific International Inc.	14-400-126
Hermle Centrifuge, Model Z 206 A	Hermle Labortechnik GmbH
Solid phase reaction vessel, 10 mL	Torviq	SF-1000
Pressure caps for reaction vessels	Torviq	PC-SF
Syringe filters, pore size 0.2 μm	Fisher Scientific Inc.	03-391-3B
Syringe filters, pore size 0.45 μm	Fisher Scientific Inc.	03-391-3A
Polypropylene centrifuge tuges, 50 mL	VWR International, LLC.	490001-626
Polypropylene centrifuge tuges, 15 mL	VWR International, LLC.	490001-620
ChemBioDraw, Ultra, Version 12.0	CambridgeSoft Corporation		CambridgeSoft is now part of PerkinElmer Inc.
Styrofoam cup, 12 Oz	Common Supermarket
Rink amide resin	Chem-Impex International, Inc.	10619
Piperidine	Chem-Impex International, Inc.	02351	Highly toxic
N, N’-diisopropylcarbodiimide	Chem-Impex International, Inc.	00110	Highly toxic
Bromoacetic acid	Chem-Impex International, Inc.	26843	Highly toxic
2-Phenylethylamine	VWR International, LLC.	EM8.07334.0250
2-Methyoxyethylamine	Sigma-Aldrich Co. LLC.	241067
N-Boc-ethylenediamine	VWR International, LLC.	AAAL19947-06
Acetic anhydride	Sigma-Aldrich Co. LLC.	252845
N, N-dimethylformamide	VWR International, LLC.	BDH1117-4LG	Further distillation before use
N, N-diisopropylethylamine	Chem-Impex International, Inc.	00141
Triisopropylsilane	Chem-Impex International, Inc.	01966
Trifluoroacetic acid	Chem-Impex International, Inc.	00289	Highly toxic
Millipore MILLI-Q Academic Water Purification System	Millipore Corporation	ZMQP60001	For generating HPLC grade water
HPLC-grade Water			Produced from Millipore MILLI-Q® Academic Water Purification System
Methanol	Pharmco-Aaper	339USP/NF	HPLC grade
Acetonitrile	Fisher Scientific International, Inc.	A998-4	HPLC grade
Diethyl ether	VWR International, LLC.	BDH1121-19L	Further distillation before use
Dichloromethane	VWR International, LLC.	BDH1113-19L	Further distillation before use
Nitrogen gas	Fresno Oxygen/Barnes Supply	NIT 50-C-F	Ultra high purity, 99.9995%
Argon gas	Fresno Oxygen/Barnes Supply	ARG 50-C-F	Ultra high purity, 99.9995%