En protokol til syntese af en 1,2-dithiolane ændrede peptid og karakterisering af Supramolekylær strukturer som følge af peptid samlesæt.
Denne rapport fokuserer på syntesen af en N-terminus 1,2-dithiolane ændret selvsamlende peptid og karakterisering af den deraf følgende samlet selvstændige Supramolekylær strukturer. Den syntesevejen udnytter faststadie syntese med den på harpiks kobling af dithiolane forløber molekyle, 3-(acetylthio) -2-(acetylthiomethyl) propanoic syre, og mikrobølgeovn-assisteret thioacetate deprotection af peptid N-terminus før endelige kavalergang fra harpiks til at give 1,2-dithiolane ændrede peptid. Efter en high-performance væskekromatografi (HPLC) rensning af 1,2-dithiolane peptid, stammer fra den kernedannende kerne af Aβ peptid forbundet med Alzheimers sygdom, er peptid vist at selv samle til cross-β amyloid fibre. Protokoller til at karakterisere den amyloid fibre ved Fouriertransformation infrarød spektroskopi (FT-IR), cirkulære dichroism spektroskopi (CD) og transmissions elektronmikroskopi (TEM) er præsenteret. Metoder til ændring af N-terminal med en 1,2-dithiolane gruppe godt karakteriseret selvsamlende peptider kan nu undersøges som modelsystemer at udvikle strategier, efter montering modifikation og udforske dynamisk kovalente kemi på Supramolekylær kemi peptid nanofiber overflader.
Den robuste peptidbinding danner kemi involveret faststadie peptidsyntese og evnen til at kontrollere rækkefølgen længde og sammensætning gør de peptider, der selv samler til Supramolekylær strukturer et stærkt undersøgte felt. De faktorer, der styrer og stabilisere peptid selvsamlede strukturer, herunder sidekæde sterisk og elektrostatiske interaktion, hydrogenbindende og hydrofobe virkninger1, tjene som et sæt af designregler. Som forskningen i disse grundlæggende designregler fortsat at gøre fremskridt, indebærer det logiske næste skridt i peptid samlesæt udvide mangfoldighed af peptid-baserede strukturer og funktioner. Mens du selv samle peptider er en alsidig biomateriale, som har været brugt i mange biomedicinske anvendelser af tuning peptid sekvens eller forsamling betingelser2,3,4, udviklingen af strategier for efter samling ændringer til peptid nanofibers5,6,7,8,9 er fortsat et relativt uudforsket område.
Dynamisk disulfid exchange og thiol kemi på overfladen af Supramolekylær strukturer er et område, der har potentiale til at give nye og funktionelle biomaterialer. Indarbejdelse af 1,2-dithiolane fraspaltning (almindeligvis et derivat af lipoinsyre (la) eller asparagusic syre (aa)) er blevet rapporteret i Liposom systemer10,11, blok copolymerer af12,13, og som organisere ankre på overflader14,15. Heri, rapporterer vi syntese og karakterisering af en selvsamlende peptid stammer fra den kernedannende kerne af Aβ peptid forbundet med Alzheimers sygdom, er ændret på N-terminus med en 1,2-dithiolane funktionelle gruppe16, 17. De resulterende Supramolekylær fibre nu fungere som en eksperimenterende platform til at studere disulfid-udveksling og thiol reaktiviteten på Supramolekylær overfladen af amyloid fibre18.
Denne artikel omhandler detaljerne i både syntesen og rensning af en N-terminale 1,2-dithiolane ændret selvsamlende peptid og karakterisering af de resulterende Supramolekylær strukturer. Syntesen af 1,2-dithiolane peptid rapporteret her har fordele, herunder en et-trins syntese for at producere dithiolane forløber, 3-(acetylthio) -2-(acetylthiomethyl) propanoic syre, og harpiksen mikroovn deprotection reaktion af de forløber thioacetate beskytte gruppe for at give den oxiderede 1,2-dithiolane gruppe udnytter ammoni…
The authors have nothing to disclose.
Forfatterne vil gerne takke Dr. B. Ellen Scanley for hendes teknisk uddannelse og hjælp ved hjælp af TEM på Connecticut State Colleges og Universitet (CSCU) Center for nanoteknologi og Dr. Ishita Mukerji på Wesleyan University for adgang til hendes CD Spektrofotometer. Arbejdet rapporteret blev delvis støttet af Science Institute på Fairfield Universitet, NASA Connecticut plads Grant Consortium, og National Science Foundation under tilskud antal CHE-1624774.
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. |