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Abstract

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Protocol

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Acknowledgements

Materials

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Chemistry

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

Published: August 20th, 2018

DOI:

10.3791/58135

1Department of Chemistry and Biochemistry, Fairfield University

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 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.

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....

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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.......

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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.......

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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 .......

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The authors would like to thank Dr. B. Ellen Scanley for her technical training and help using the TEM at the Connecticut State Colleges and University (CSCU) Center for Nanotechnology and Dr. Ishita Mukerji at Wesleyan University for access to her CD spectrophotometer. The work reported was in part supported by the Science Institute at Fairfield University, the NASA Connecticut Space Grant Consortium, and by the National Science Foundation under Grant Number CHE-1624774.

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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.

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