Sintesi e caratterizzazione di 1,2-Dithiolane per volta peptidi autoassemblanti
Summary
Un protocollo per la sintesi di un 1,2-dithiolane modificato peptide e la caratterizzazione delle strutture supramolecolari derivanti dal peptide self-assembly.
Abstract
Questo rapporto si concentra sulla sintesi di un N-terminale 1,2-dithiolane modificate autoassemblanti peptide e la caratterizzazione del risultante self-assembled strutture supramolecolari. Il percorso sintetico si avvale della sintesi del peptide di fase solida con l’accoppiamento su resina della molecola del precursore dithiolane, 3-(acetylthio) -2-acido propanoico (acetylthiomethyl) e il Tio acetato assistita da microonde deprotection del peptide N-terminale prima scissione finale dalla resina di cedere l’1,2-dithiolane per volta peptide. Dopo la purificazione di cromatografia liquida ad alte prestazioni (HPLC) del peptide 1,2-dithiolane, derivato dal nucleo nucleanti del peptide Aβ associato a malattia di Alzheimer, il peptide è indicato per assemblarsi in fibre amiloidi Croce-β. Protocolli per caratterizzare le fibre amiloidi-trasformata di Fourier spettroscopia infrarossa (FT-IR), spettroscopia di dicroismo circolare (CD) e microscopia elettronica a trasmissione (TEM) sono presentati. I metodi di modificazione del N-terminale con una molecola di 1,2-dithiolane a peptidi autoassemblanti ben caratterizzati ora possono essere esplorati come sistemi modello per sviluppare strategie di modificazione post-montaggio e dinamica chimica covalente e dintorni superfici di nanofibra peptide supramolecolari.
Introduction
Il legame peptidico robusto formando chimica coinvolta nella sintesi del peptide di fase solida e la capacità di controllare la composizione e la lunghezza della sequenza rendono i peptidi che auto-assemblarsi in strutture supramolecolari un campo fortemente ricercato. I fattori che controllano e stabilizzano strutture auto-assemblate del peptide, tra cui la catena laterale sterico e interazioni elettrostatiche, legame idrogeno e idrofobo effetti1, servono come un insieme di regole di progettazione. Come la ricerca in queste regole fondamentali di progettazione continua a progredire, il logico passo successivo nel peptide coinvolge auto-assemblaggio sta espandendo la diversità delle funzioni e strutture basate su peptidi. Durante l’auto-assemblaggio di peptidi sono un biomateriale versatile che sono stati utilizzati per molte applicazioni biomediche sintonizzando il peptide sequenza o assieme condizioni2,3,4, lo sviluppo di strategie per modifiche post-montaggio al peptide nanofibre5,6,7,8,9 rimane un’area relativamente inesplorata.
Chimica di cambio e del tiolo bisolfuro dinamico alla superficie delle strutture supramolecolari è un’area che ha il potenziale per produrre biomateriali nuovi e funzionale. L’incorporazione di 1,2-dithiolane moiety (comunemente un derivato di acido lipoico (la) o acido asparagusic (aa)) sono stati segnalati in liposomi sistemi10,11, blocco copolimeri12,13e come organizzazione di ancoraggi alle superfici14,15. Qui, segnaliamo la sintesi e la caratterizzazione di un peptide autoassemblanti derivato dal nucleo nucleanti del peptide Aβ associato con la malattia di Alzheimer che è stata modificata al N-terminale con un gruppo funzionale di 1,2-dithiolane16, 17. Le fibre risultanti supramolecolari ora servono come piattaforma sperimentale per lo studio della reattività di bisolfuro-cambio e tiolo alla superficie supramolecolare di fibre amiloidi18.
Protocol
Representative Results
Discussion
Questo articolo discute i dettagli sia la sintesi e la purificazione di un peptide di autoassemblanti 1,2-dithiolane per volta N-terminale e la caratterizzazione delle strutture supramolecolari risultante. La sintesi del peptide 1,2-dithiolane segnalato qui presenta i vantaggi, tra cui una sintesi di uno stadio per produrre il precursore dithiolane, 3-(acetylthio) -2-acido propanoico (acetylthiomethyl) e la resina il forno a microonde deprotezione reazione dei Tio acetato precursore protezione gruppo per produrre la fraz…
Declarações
The authors have nothing to disclose.
Acknowledgements
Gli autori piacerebbe ringraziare il Dr. B. Ellen Scanley per la sua formazione tecnica e aiuto utilizzando il TEM al Connecticut State Colleges e Università (CSCU) centro per le nanotecnologie e Dr. Ishita Mukerji alla Wesleyan University per l’accesso al suo CD spettrofotometro. Il lavoro segnalato è stata in parte sostenuto dall’Istituto scienza alla Fairfield University, la NASA Connecticut Space Grant Consortium e dalla National Science Foundation sotto Grant numero-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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