Synthese und Charakterisierung von 1,2-Dithiolane geändert selbstorganisierende Peptide
Summary
Ein Protokoll für die Synthese von 1,2-Dithiolane modifizierte Peptid und die Charakterisierung der supramolekularen Strukturen durch das Peptid Selbstmontage.
Abstract
Dieser Bericht konzentriert sich auf die Synthese von 1,2-Dithiolane ein N-Terminus selbstorganisierende Peptid und die Charakterisierung der resultierenden geändert selbst montiert supramolekulare Strukturen. Der Syntheseweg nutzt der Festphasen-Peptidsynthese mit der auf-Harz-Kopplung von Dithiolane Vorläufer Molekül, 3-(Acetylthio) -2-(Acetylthiomethyl) Propanoic Säure und die Mikrowellen-gestützte Thioacetate deprotection des Peptids N-Terminus vor dem endgültigen Spaltung aus dem Harz der 1,2-Dithiolane Ausbeute geändert Peptid. Nach der Hochleistungs-Flüssigkeitschromatographie (HPLC) Reinigung des 1,2-Dithiolane Peptids, abgeleitet aus dem nukleierenden Kern der Aβ-Peptid verbunden mit Krankheit Alzheimers zeigt das Peptid in Kreuz-β-Amyloid-Fasern selbst zusammensetzen. Protokolle zur Charakterisierung der Amyloid Fasern von Fourier-Transform-Infrarot-Spektroskopie (FT-IR), kreisförmigen Dichroismus Spektroskopie (CD) und Transmissionselektronenmikroskopie (TEM) werden vorgestellt. Die Methoden von N-terminale Anpassung mit einem 1,2-Dithiolane Glyko-gut charakterisierten selbstorganisierende Peptide können jetzt erkundet werden, als Modellsysteme, Post-Montage Modifikation Strategien entwickeln und dynamische kovalente Chemie zu erkunden Supramolekulare Peptid Nanofaser Oberflächen.
Introduction
Die robuste Peptidbindung bilden Chemie beteiligt Festphasen-Peptidsynthese und die Fähigkeit zur Steuerung der Reihenfolge Länge und Zusammensetzung machen die Peptide, die in supramolekularen Strukturen stark erforschten Bereich selbst zusammensetzen. Die Faktoren, die zu kontrollieren und zu stabilisieren, Peptid selbst-zusammengebauten Strukturen, einschließlich Side-Chain sterische und elektrostatische Wechselwirkungen, Wasserstoff-Bindung und hydrophobe Effekte1, dienen als eine Reihe von Design-Regeln. Wie die Forschung zu diesen grundlegenden Designregeln schreitet weiter voran, geht der nächste logische Schritt in Peptid Selbstmontage erweitert die Vielfalt der Peptid-basierte Strukturen und Funktionen. Während der Montage selbst Peptide sind vielseitige Biomaterial, das für viele biomedizinische Anwendungen herangezogen wurden, durch tuning die Peptid-Sequenz oder einer Baugruppe Bedingungen2,3,4, die Entwicklung von Strategien für nach dem Zusammenbau Änderungen an Peptid Nanofasern5,6,7,8,9 bleibt ein relativ unerforschten Bereich.
Dynamische Disulfid-Austausch und Thiol-Chemie an der Oberfläche der supramolekularen Strukturen ist ein Bereich, der das Potenzial hat, neuen und funktionalen Biomaterialien ergeben. Die Einbeziehung von 1,2-Dithiolane Moieties (häufig ein Derivat von Liponsäure (la) oder asparagusic Säure (aa)) gemeldet wurden, in Liposomen Systeme10,11, Block-Copolymere12,13, sowie Organisation von Ankern an Oberflächen14,15. Hier berichten wir über die Synthese und Charakterisierung eines selbstorganisierenden Peptids, abgeleitet aus dem nukleierenden Kern der Aβ-Peptid Zusammenhang mit Alzheimer-Krankheit, die am N-Terminus mit einem 1,2-Dithiolane funktionelle Gruppe16geändert wird, 17. Die daraus resultierenden supramolekularen Fasern dienen nun als eine experimentelle Plattform die Disulfid-Austausch und Thiol Reaktivität an der supramolekularen Oberfläche des Amyloid Fasern18zu studieren.
Protocol
Representative Results
Discussion
Dieser Artikel beschreibt die Details der Synthese und Reinigung von einer N-terminalen 1,2-Dithiolane geändert selbstorganisierende Peptid und die Charakterisierung der daraus resultierenden supramolekularen Strukturen. Die Synthese von 1,2-Dithiolane Peptids hier berichtet hat die Vorteile, einschließlich einer einstufigen Synthesis um Dithiolane Vorstufe, 3-(Acetylthio)-2 zu produzieren-Propanoic Säure (Acetylthiomethyl) und das Harz Mikrowelle Deprotection Reaktion der Vorläufer Thioacetate Schutz der Gruppe, um …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Die Autoren möchten Danke Dr. B. Ellen Scanley für ihre technische Ausbildung und hilf mit die TEM an der Connecticut State Colleges und University (CSCU) Zentrum für Nanotechnologie und Dr. Ishita Mukerji an der Wesleyan University für den Zugriff auf ihre CD Spektrophotometer. Die Arbeit berichtet wurde zum Teil durch das Science Institute an der Fairfield University, die NASA Connecticut Space Grant-Konsortium und der National Science Foundation unter Grant Anzahl CHE-1624774 unterstützt.
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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