Syntese og karakterisering av 1,2-Dithiolane endret selv av peptider
Summary
En protokoll for syntesen av en 1,2-dithiolane endret peptid og karakterisering av supramolecular strukturer som følge av peptid selvtillit forsamlingen.
Abstract
Rapporten fokuserer på syntesen av en N-terminus 1,2-dithiolane endret selv montasje peptid og karakterisering av den resulterende samlet selv supramolecular strukturer. Syntetisk ruten tar nytte av solid-fase peptid syntese med på harpiks koblingen av dithiolane Forløperen molekylet, 3-(acetylthio) -2-(acetylthiomethyl) propanoic syre og mikrobølgeovn-assistert thioacetate deprotection av peptid N-terminus før siste cleavage fra harpiks til 1,2-dithiolane endret peptid. Etter høy ytelse flytende kromatografi (HPLC) rensing av 1,2-dithiolane peptid, avledet fra nucleating kjernen i Aβ peptid forbundet med Alzheimers sykdom, vises peptid å selv samle til kryss-β amyloid fiber. Protokoller som karakteriserer amyloid fiber av Fourier-transform infrarød spektroskopi (FT-IR), sirkulære dichroism spektroskopi (CD) og overføring elektronmikroskop (TEM) presenteres. Metoder for N-terminal endring med en 1,2-dithiolane moiety godt karakterisert selv av peptider kan nå bli utforsket som modellsystemer å utvikle etter montering endring strategier og utforske dynamiske kovalente kjemi på supramolecular peptid nanofiber overflater.
Introduction
Robust peptid bond danner kjemi involvert i solid-fase peptid syntese og kontrollere rekkefølgen lengde og komposisjon gjøre peptidene som selv samle inn supramolecular strukturer et tungt undersøkt felt. Faktorene som kontrollere og stabilisere peptid selv montert strukturer, inkludert side kjede steric og elektrostatiske interaksjoner, hydrogenbinding og hydrofobe effekter1, tjene som et sett med utformingsregler. Som forskningen på disse grunnleggende regler fortsetter å gå videre, innebærer det logiske neste skrittet i peptid selvtillit forsamlingen utvide mangfoldet av peptid-baserte strukturer og funksjoner. Mens selv monterer peptider er en allsidig biomateriale som har blitt brukt for mange biomedisinsk programmer av tuning peptid sekvens eller montering forhold2,3,4, utvikling av strategier for etter montering endringer peptid nanofibers5,6,7,8,9 fortsatt en relativt uutforsket område.
Dynamisk disulfide exchange og thiol kjemi på overflaten av supramolecular strukturer er et område som har potensial til å gi ny og funksjonell biologisk materiale. Inkorporering av 1,2-dithiolane moieties (vanligvis et derivat av lipoic syre (la) eller asparagusic syre (aa)) er rapportert i liposome systemer10,11, blokk copolymers12,13, og som organisere ankere overflater14,15. Her, rapportere vi syntese og karakterisering av en selvstendig montering peptid avledet fra nucleating kjernen i Aβ peptid forbundet med Alzheimers sykdom som er endret på N-terminus med en 1,2-dithiolane funksjonsgruppe16, 17. Den resulterende supramolecular fiber nå fungere som en eksperimentell plattform å studere disulfide-utveksling og thiol reaktivitet på supramolecular overflaten av amyloid fiber18.
Protocol
Representative Results
Discussion
Denne artikkelen diskuterer detaljer om både syntese og rensing av en N-terminal 1,2-dithiolane endret selv montasje peptid og karakterisering av de resulterende supramolecular strukturene. Syntese av 1,2-dithiolane peptid rapporterte her har fordeler, inkludert en ettrinns syntese å produsere dithiolane forløperen, 3-(acetylthio) -2-(acetylthiomethyl) propanoic syre, og på-harpiks mikrobølgeovn deprotection reaksjon av det forløperen thioacetate beskytte gruppe å gi oksidert 1,2-dithiolane moiety utnytte ammonium…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil takke Dr. B. Ellen Scanley for hennes teknisk opplæring og hjelp til TEM på Connecticut statlige høgskoler og University (CSCU) senter for nanoteknologi og Dr. Ishita Mukerji ved Wesleyan University for tilgang til hennes CD spektrofotometeret. Arbeidet som er rapportert ble delvis støttet av Science Institute ved Fairfield University, NASA Connecticut plass Grant konsortiet, og 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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