Syntes och karakterisering av 1,2-Dithiolane modifierade själv montering peptider
Summary
Ett protokoll för syntesen av en 1,2-dithiolane modifierade peptid och karakterisering av supramolekylär strukturer som härrör från peptiden självmontering.
Abstract
Denna rapport fokuserar på syntesen av en N-terminalen 1,2-dithiolane modifierade själv montering peptid och karakterisering av den resulterande monterade själv supramolekylär strukturer. Syntetiska rutten tar fördel av fasta fasen peptidsyntesen med på-harts kopplingen av dithiolane föregångaren molekyl, 3-(acetylthio) -2-(acetylthiomethyl) propansyra syra och den mikrovågsugn-assisted thioacetate deprotection av peptiden N-terminalen innan slutlig klyvning från kådan ge den 1,2-dithiolane modified peptid. Efter högpresterande vätskekromatografi (HPLC) rening av 1,2-dithiolane peptiden, härrör från nucleating kärnan av Aβ peptiden är associerad med Alzheimers sjukdom, visas peptiden att själv montera in cross-β amyloid fibrerna. Protokoll att karakterisera amyloid fibrerna av Fourier-transform infraröd spektroskopi (FT-IR), cirkulärdichroism spektroskopi (CD) och transmissionselektronmikroskopi (TEM) presenteras. Metoderna för N-terminala modifiering med en 1,2-dithiolane del till välkarakteriserad själv montering peptider kan nu utforskas som modellsystem att utveckla efter montering modifiering strategier och utforska dynamiska kovalent kemi på supramolekylär peptid nanofiber ytor.
Introduction
Den robusta peptidbindning bildas kemi deltar i fasta fasen peptidsyntesen och möjligheten att styra sekvensen längd och sammansättning gör de peptider som själv monterar i supramolekylär strukturer ett kraftigt utforskade fält. De faktorer som styr och stabilisera peptid själv monterade strukturer, däribland sidokedjan sterisk och elektrostatisk interaktioner, väte bindning och hydrofoba effekter1, fungera som en uppsättning designregler. Som forskningen om dessa grundläggande designregler fortsätter att göra framsteg, innefattar det logiska nästa steget i peptid självmontering utöka mångfalden av peptid-baserad strukturer och funktioner. Samtidigt självmonterande peptider är en mångsidig biomaterial som har använts för många biomedicinska tillämpningar av tuning peptid sekvens eller församlingen villkor2,3,4, utveckling av strategier för efter montering ändringar peptid nanofibrer5,6,7,8,9 är fortfarande ett relativt outforskat område.
Dynamiska disulfid utbyte och thiol kemi vid ytan av supramolekylär strukturer är ett område som har potential att ge nya och funktionella biomaterial. Införlivandet av 1,2-dithiolane beståndsdelarna (vanligen ett derivat av liponsyra (la) eller asparagusic syra (aa)) har rapporterats i Liposom system10,11, block sampolymerer12,13, och som organisera ankare ytor14,15. Häri, rapporterar vi syntes och karakterisering av en egen montering peptid som härrör från nucleating kärnan av Aβ peptiden är associerad med Alzheimers sjukdom som ändras vid N-terminalen med en 1,2-dithiolane funktionell grupp16, 17. Den resulterande Supramolekylära fibrerna nu fungera som en experimentell plattform att studera disulfid-exchange och thiol reaktiviteten på supramolekylär ytan av amyloid fibrer18.
Protocol
Representative Results
Discussion
Artikeln detaljerna i både syntes och rening av en N-terminal 1,2-dithiolane modified själv montering peptid och karakterisering av de resulterande Supramolekylära strukturerna. Syntesen av 1,2-dithiolane peptiden redovisas här har fördelar, inklusive en one-step syntes för att producera dithiolane föregångare, 3-(acetylthio) -2-(acetylthiomethyl) propansyra syra och på-kådan mikrovågsugn deprotection reaktion av den föregångaren thioacetate skydda gruppen för att ge den oxiderade 1,2-dithiolane biexponenti…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Författarna vill tacka Dr B. Ellen Scanley för hennes teknisk utbildning och hjälp med TEM vid Connecticut State Colleges och universitet (CSCU) Center för nanoteknologi och Dr Ishita Mukerji vid Wesleyan University för tillgång till hennes CD spektrofotometer. Det arbete som rapporterats var delvis stöds av den Science Institute vid Fairfield University, NASA Connecticut utrymme bevilja konsortiet, och National Science Foundation under Grant nummer 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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