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

Summary

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.

Abstract

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 Aβ peptide associated with Alzheimer's disease, the peptide is shown to self-assemble into cross-β 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.

Introduction

The robust peptide bond forming chemistry involved in solid-phase peptide synthesis and the ability to control sequence length and composition make the peptides that self-assemble into supramolecular structures a heavily researched field. The factors that control and stabilize peptide self-assembled structures, including side chain steric and electrostatic interactions, hydrogen bonding, and hydrophobic effects¹, serve as a set of design rules. As the research into these fundamental design rules continues to progress, the logical next step in peptide self-assembly involves expanding the diversity of peptide-based structures and functions. While....

Protocol

1. Synthesis and Purification of 1,2-Dithiolane Modified Peptide

1. Synthesis of dithiolane precursor, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid¹⁹.
   1. Add 1 g of 3-bromo-2-(bromomethyl)propionic acid (1 equiv.) dissolved in minimal amount of 1 M NaOH (approximately 4 mL) to a 25-mL round bottom reaction flask with stirring at 55 °C. Seal the reaction flask with a septa and place under nitrogen atmosphere.
   2. Prepare a solution containing 1.49 g of potassium thioacetate (3.2 equiv.) in 4 mL of deionized water and 3 mL of 2 M sulfuric acid (H₂SO₄) to create thioacetic a....

Results

Aside from the initial one-step synthesis of the dithiolane precursor molecule, the rest of the 1,2-dithiolane modified peptide synthesis occurs on solid support (Figure 1A). The conversion of 3-bromo-2-(bromomethyl)propionic acid to 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, the dithiolane precursor, is confirmed by ¹H and ¹³C NMR (Figure 1B and C) before it is coupled to the free N.......

Discussion

This article discusses the details of both the synthesis and the purification of an N-terminal 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting supramolecular structures. The synthesis of the 1,2-dithiolane peptide reported here has the advantages, including a one-step synthesis to produce the dithiolane precursor, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the on-resin microwave deprotection reaction of the precursor thioacetate protecting group to yield the oxidized .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rink amide MBHA resin, high load	Gyros Protein Technologies	RAM-5-HL	Avoid contact with skin and eyes; do not inhale
N,N-Dimethylformamide	Fisher Scientific	D119-4	Flammable liquid and vapor; irritating to eyes and skin; Use personal protective equipment; keep away from open flame
Fmoc-L-Val-OH	Gyros Protein Technologies	FLA-25-V	Wear personal protective equipment; do not inhale
Fmoc-L-Leu-OH	Gyros Protein Technologies	FLA-25-L	Wear personal protective equipment; do not inhale
Fmoc-L-Lys(Boc)-OH	Gyros Protein Technologies	FLA-25-KBC	Wear personal protective equipment; do not inhale
Fmoc-L-Phe-OH	Gyros Protein Technologies	FLA-25-F	Wear personal protective equipment; do not inhale
Fmoc-L-Ala-OH	Gyros Protein Technologies	FLA-25-A	Wear personal protective equipment; do not inhale
Fmoc-L-Gln(Trt)-OH	Gyros Protein Technologies	FLA-25-QT	Wear personal protective equipment; do not inhale
N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate	Gyros Protein Technologies	26432	Causes skin, eye and respiratory irritation; do not inhale; use under hood or in well ventilated area
0.4 M N-methylmorpholine in DMF	Gyros Protein Technologies	PS3-MM-L	highly flammable; wear personal protective equipment; keep away from heat and keep container tightly closed; do not inhale or swallow; wash skin thoroughly after handling
20% piperidine in DMF	Gyros Protein Technologies	PS3-PPR-L	Causes severe eye and skin burns; Flammable Liquid and vapor; Do not inhale
dichloromethane	Fisher Scientific	D37-4	May cause cancer; Do not inhale; Wear personal protective equipment; use under hood only; if contacted rise with water for at least 15 minutes and obtain medical attention
acetonitrile	Fisher Scientific	A998-4	Flammable; irritating to eyes; Use personal protective equipment; Use only under a fume hood; keep away from open flame or hot surface; if contacted rinse wiith water for at least 15 minutes and obtain medical attention
trifluoroacetic acid	Fisher Scientific	A116-50	Causes severe burns; do not inhale; harmful to aquatic life; use personal protective equipment; use only under fume hood; if contacted rinse with water for at least 15 minutes and obain immediate medical attention
4% uranyl acetate	Electron Microscopy Sciences	22400-4	Do not inhale; harmful to aquatic life
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid	Acros Organics	AC172571000	Do not inhale; use outdoors or in well-ventilated area
nitrogen Gas	TechAir		Contents under pressure, may explode if heated
3-bromo-2-(bromomethyl)propionic acid	Alfa Aesar	AAA1963014	Do not inhale; causes irritation to skin and eyes; corrosive
sodium hydroxide	Fisher Scientific	S318-100	Use personal protective equipment; use only under fume hood; if contact rinse area for at least 15 minutes and obtain medical attention
potassium thioacetate	Acros Organics	AC221300250	Causes skin and eye irritation; do not inhale; use personal protective equipment
sulfuric acid	Fisher Scientific	SA213	Causes burns; keep away from water; keep away from combustible material; do not inhale; use personal protective equipment; if contact rinse area for at least 15 minutes and obtain medical attention
chloroform-d	Acros Organics	AC320690075	Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention
chloroform	Fisher Scientific	C298-4	Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention
N,N-diisopropylethylamine	Acros Organics	AC367841000	Highly flammable; harmful to aquatic life; wear personal protective equipment; do not swallow
ammonium hydroxide	Fisher Scientific	A669S-500	Corrosive; do not inhale
methanol	Fisher Scientific	A452-4	Flammable liquid and vapor; use personal protective equipment; do not inhale; If contact rinse area for at least 15 minutes and obtain medical attention
triisopropylsilane	Sigma Aldrich	233781	Flammable; use personal proctective safety equipment; keep container tightly closed
diethyl ether	Fisher Scientific	E138-1	Extremely flammable; Irritating to skin and eyes; Use personal protective equipment
2,5-dihydroxybenzoic acid	Sigma Aldrich	39319-10x10MG-F	do not inhale; irritating to skin and eyes
alpha-cyano-4-hydroxycinnamic acid	Alfa Aesar	AAJ67635EXK
c18 zip-tip	Millipore	ZTC18S096
tris(2-carboxyethyl) phospine hydrochloride	Thermo Scientific	PI20490
silica gel 60 F254 coated aluminum-backed TLC sheets	EMD Millipore	1.05549.0001
Thin walled Precision NMR tubes	Bel-Art	663000585	5mm O.D.
All-plastic Norm-Ject syringes	Air Tite	AL10
single-use needle	BD PrecisionGlide	BD 305185	used needles get disposed on in sharps waste container
disposable fritted syringe	Torviq	SF1000LL	10mL fritted syringes were used in the report, but larger syringes are avaibale if needed for larger scale synthesis.
carbon grid	Ted Pella, Inc.	CF200-CU	Make sure to prepare samples and staining on the carbon grid side, not the shiny copper side of grid
self-closing tweezers	Electron Microscopy Sciences	78318-3X	very sharp tips, length: 120 mm
0.1 mm short path length cell	Starna Cells, Inc.	20/C-Q-0.1	Fragile
10mL Vessel Caps	CEM	909210
10mL Pressure Vessels	CEM	908035
Aeris Semi-Prep HPLC column	Phenomenex	00F-4632-N0	150 x 10mm
cell holder	Starna Cells, Inc.	CH-2049	Needed when using short pathlength cells
PS3 peptide synthesizer	Gyros Protein Technologies
DiscoverSP Microwave Reactor	CEM
centrifuge	HERMLE	Z 206 A	used a fixed 6x50 mL rotor
HPLC	Shimadzu		UV Detector
nuclear magnetic resonance spectrometer	Avance, Bruker		300 MHz
MALDI-TOF mass spectrometer	Axima Confidence, Shimadzu
lyophilizer	Millrock Technology	BT85A
Fourier-Transform Infrared Spectrometer	Alpha Tensor, Bruker
Transmission Electron Microscope	Tecnai Spirit, FEI		Used with Gatan Orius Fiberoptic CCD digital camera. Accessed at CSCU Center for Nanotechnology
Circular Dichroism Spectropolarimeter	J-810, JASCO		Used with a six-cell Peltier temperature controller. Accessed at Wesleyan University.