Synthèse et caractérisation de 1, 2-Dithiolane modifiée Peptides auto-assemblage
Summary
Un protocole pour la synthèse d’un 1, 2-dithiolane modifiée peptide et la caractérisation des structures supramoléculaires résultant de la peptide auto-assemblage.
Abstract
Ce rapport met l’accent sur la synthèse d’un N-terminal 1, 2-dithiolane modifiée auto-assemblage de peptide et la caractérisation du résultant self-assembled structures supramoléculaires. La voie de synthèse profite de la synthèse de peptide de phase solide avec l’accouplement sur résine de la molécule précurseur dithiolane, 3-(acetylthio) -2-acide propanoïque (acetylthiomethyl) et le thioacétate assistée par micro-ondes deprotection du peptide N-terminale avant dernier clivage de la résine pour donner le 1, 2-dithiolane modifiée peptide. Après la purification de la chromatographie en phase liquide à haute performance (HPLC) du peptide 1, 2-dithiolane, dérivé de la nucléation core du peptide Aβ associée à la maladie d’Alzheimer, le peptide est avéré s’auto-assembler en fibres amyloïdes Croix-β. Protocoles pour caractériser les fibres amyloïdes par spectroscopie infrarouge à transformée de Fourier (FT-IR), spectroscopie de dichroïsme circulaire (CD) et microscopie électronique à transmission (TEM) sont présentés. Les méthodes de modification de la N-terminale avec une portion de 1, 2-dithiolane aux peptides auto-assemblage bien caractérisés peuvent maintenant être explorées comme systèmes modèles pour élaborer des stratégies de modification après montage et Explorer chimie covalente dynamique sur surfaces de peptide supramoléculaire de nanofibres.
Introduction
La liaison peptidique robuste formant chimie impliquée dans la synthèse de peptide de phase solide et la possibilité de contrôler la composition et la durée d’une séquence font les peptides qui s’auto-assembler en structures supramoléculaires un champ largement documenté. Les facteurs qui contrôlent et stabilisent les structures auto-assemblées peptide, y compris la chaîne latérale stérique et interactions électrostatiques, des liaisons hydrogènes et effets hydrophobes1, constituent un ensemble de règles de conception. Comme des recherches menées sur ces règles de conception fondamentale continuent de progresser, l’étape logique suivante en peptide auto-assemblage consiste à élargir la diversité des fonctions et des structures à base de peptides. Lors de l’assemblage des peptides sont un biomatériau polyvalent qui ont été utilisés pour de nombreuses applications biomédicales en syntonisant le peptide séquence ou montage conditions2,3,4, l’élaboration de stratégies pour modifications après montage peptide nanofibres5,6,7,8,9 reste un domaine relativement inexploré.
Disulfure dynamique échange thiol chimie et à la surface des structures supramoléculaires est un secteur qui a le potentiel d’engendrer des biomatériaux nouveaux et fonctionnels. L’incorporation des portions de 1, 2-dithiolane (généralement un dérivé de l’acide lipoïque (la) ou l’acide asparagusique (aa)) ont été signalés dans un liposome systèmes10,11, copolymères de bloc12,13et en tant que ancres à surfaces14,15de l’organisation. Ici, nous rapportons la synthèse et la caractérisation d’un auto-assemblage peptide dérivé de la nucléation core du peptide Aβ associée à la maladie d’Alzheimer qui est modifiée à l’extrémité N-terminale avec un 1, 2-dithiolane groupe fonctionnel16, 17. Les fibres qui en résulte supramoléculaires servent désormais une plateforme expérimentale pour étudier la réactivité disulfure intracirconscriptions et thiols en surface des fibres amyloïdes18supramoléculaire.
Protocol
Representative Results
Discussion
Cet article explique les détails de la synthèse et la purification d’un peptide auto-assemblage de N-terminal 1, 2-dithiolane modifiée et la caractérisation des structures supramoléculaires qui en résulte. La synthèse du peptide 1, 2-dithiolane rapporté ici a des avantages, y compris une synthèse en une étape pour produire le précurseur dithiolane, 3-(acetylthio) -2-acide propanoïque (acetylthiomethyl) et la résine sur micro-ondes réaction de déprotection de la précurseur thioacétate de groupe pour obt…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Les auteurs tiennent à remercier Dr B. Ellen Scanley pour sa formation technique et aider grâce à la TEM au Connecticut State Colleges and University (CSCU) Center for Nanotechnology et Dr Ishita Mukerji à la Wesleyan University pour son CD spectrophotomètre. Le travail a été en partie pris en charge par l’Institut des sciences à l’Université de Fairfield, la NASA Connecticut Space Grant Consortium et par la National Science Foundation sous Grant nombre CHE-1624774.
Materials
| 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 6×50 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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