Síntese e caracterização de 1,2-Ditiolano modificado peptídeos auto-montagem
Summary
Um protocolo para a síntese de um 1,2-Ditiolano modificado de peptídeo e a caracterização das estruturas supramoleculares resultantes do peptide auto-montagem.
Abstract
Este relatório centra-se na síntese de um N-terminal 1,2-Ditiolano modificado auto-montagem de peptídeo e a caracterização dos resultante Self montadas estruturas supramoleculares. A rota sintética tira proveito da síntese do peptide de fase sólida com o engate em resina Ditiolano precursor da molécula de, 3-(acetylthio) -2-ácido (acetylthiomethyl) e o tioacetato assistida por microondas deprotection do peptide N-terminal antes de clivagem final da resina para produzir o 1,2-Ditiolano modificado do peptide. Após a purificação de cromatografia líquida de alto desempenho (HPLC) do peptide 1,2-Ditiolano, derivado do núcleo de nucleação do peptide Aβ associado com a doença de Alzheimer, o peptídeo é mostrado a auto-montagem em fibras de amiloides Cruz-β. Protocolos para caracterizar as fibras amiloides por espectroscopia de infravermelho-transformada de Fourier (FT-IR), espectroscopia de Dicroísmo circular (CD) e microscopia eletrônica de transmissão (TEM) são apresentados. Os métodos de modificação do N-terminal com um grupo de 1,2-Ditiolano bem caracterizadas péptidos auto-montagem agora podem ser explorados como sistemas modelo para desenvolver estratégias de modificação pós-montagem e explorar o dinâmica química covalente superfícies de nanofibras de peptídeo supramolecular.
Introduction
Robusta peptídica formando a química envolvida na síntese do peptide de fase sólida e a capacidade de controlar a composição e o comprimento da sequência fazer os peptídeos que auto-montagem em estruturas supramoleculares um campo fortemente pesquisado. Os fatores que controlam e estabilizar a estrutura Self montada do peptide, incluindo cadeia lateral estérica e interações eletrostáticas, ligações de hidrogênio e efeitos hidrofóbicos1, servem como um conjunto de regras de design. Como a investigação sobre essas regras de design fundamental continua a progredir, o próximo passo lógico na peptídeo auto-montagem envolve expandir a diversidade de funções e estruturas do peptide-baseado. Ao montar auto peptídeos são um biomaterial versátil que foram usados para muitas aplicações biomédicas ajustando o peptídeo sequência ou conjunto condições2,3,4, o desenvolvimento de estratégias para modificações pós-montagem do peptide nanofibras5,6,7,8,9 continua a ser uma área relativamente inexplorada.
Bissulfeto dinâmico intercâmbio e tiol química na superfície de estruturas supramoleculares é uma área que tem potencial para produzir biomateriais novos e funcionais. A incorporação de partes de 1,2-Ditiolano (comumente um derivado do ácido lipoico (la) ou asparagúsico (aa)) têm sido relatados em lipossomas sistemas10,11,12,de copolímeros bloco13e como organizando as âncoras às superfícies14,15. Neste documento, nós relatamos a síntese e caracterização de um peptídeo de auto-montagem derivado do núcleo de nucleação do peptide Aβ associado com a doença de Alzheimer que é modificada no N-terminal com um grupo funcional de 1,2-Ditiolano16, 17. As fibras supramoleculares resultantes agora servem como uma plataforma experimental para estudar a reatividade de bissulfeto-troca e tiol na superfície supramolecular de fibras amiloides18.
Protocol
Representative Results
Discussion
Este artigo discute os detalhes de tanto a síntese e a purificação de um peptídeo de auto-montagem de 1,2-Ditiolano modificado N-terminal e a caracterização das estruturas supramoleculares resultantes. A síntese do peptide 1,2-Ditiolano relatado aqui tem as vantagens, incluindo a síntese de uma etapa para produzir o precursor de Ditiolano, 3-(acetylthio) -2-ácido (acetylthiomethyl) e a resina no microondas reação de desproteção do precursor tioacetato grupo para render o moiety oxidado 1,2-Ditiolano, utiliza…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Os autores gostaria de agradecer o Dr. B. Ellen Scanley para sua formação técnica e ajudar usando a temperatura no centro da Universidade (CSCU) e faculdades do estado de Connecticut para nanotecnologia e Dr. Ishita Mukerji na Universidade wesleyana para acesso ao seu CD Espectrofotómetro. O trabalho relatado foi apoiado em parte pelo Instituto de Ciências da Universidade de Fairfield, a NASA Connecticut Space Grant Consortium e pela National Science Foundation sob Grant número-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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