1,2-Dithiolane malzemelerin ve kendi kendine montaj peptidler değiştirilme tarihi
Summary
1,2-dithiolane sentezi için bir protokol peptid ve peptid kendinden montajlı kaynaklanan supramolecular yapıları karakterizasyonu değiştiren.
Abstract
Kendi kendine montaj peptid ve sonucu olan karakterizasyonu bir N-terminus 1,2-dithiolane sentezi üzerinde duruluyor değiştiren bu rapor supramolecular yapıları kendi kendine monte. Katı faz peptid sentez dithiolane habercisi molekülü 3-(acetylthio) -2-reçine MANŞONLA sentetik rota yararlanır-(acetylthiomethyl) propanoic asit ve mikrodalga destekli thioacetate deprotection peptid N-terminus reçine üzerinden son bölünme önce 1,2-dithiolane vermeye peptid değiştiren. Alzheimer hastalığı, ilişkili Aβ peptid nucleating çekirdeğinden elde edilen 1,2-dithiolane peptid, yüksek performanslı sıvı kromatografi (HPLC) arıtma sonra peptid çapraz-β amiloid lifleri kendi kendine montajı gösterilmiştir. Fourier dönüşümü kızılötesi spektroskopi (FT-IR), dairesel dichroism spektroskopisi (CD) ve transmisyon elektron mikroskobu (TEM) amiloid lifler karakterize etmek için protokolleri mevcuttur. N-terminal geliştirmeleriyle 1,2-dithiolane yan iyi karakterize kendi kendine montaj peptidler yöntemleri şimdi sonrası derleme değişiklik stratejiler geliştirmek ve dinamik kovalent kimya üzerinde keşfetmek için model sistemleri olarak keşfedilmeyi olabilir Supramolecular peptid nanofiber yüzeyler.
Introduction
Kimya şekillendirme sağlam peptit bağı katı fazlı peptid sentezinde yer ve sıra uzunluğu ve kompozisyon kontrol yeteneği yapmak supramolecular yapılarına ağır araştırılmış bir alanı kendi kendine araya peptidler. Kontrol ve yan zincir steric dahil olmak üzere peptid kendi kendine monte yapılar ve elektrostatik etkileşimler, hidrojen bağı ve hidrofobik etkileri1, stabilize faktörler tasarım kuralları kümesi olarak hizmet vermektedir. Bu temel tasarım kuralları içine araştırma ilerlemeye devam ediyor gibi peptid mantıksal sonraki adımda kendinden montajlı çeşitliliği peptid temel yapıları ve işlevleri genişletme kapsar. Kendi kendine peptidler montaj sırasında birçok biyomedikal uygulamalarda peptit dizisi veya derleme koşullar2,3,4, için strateji geliştirme ayarlama tarafından kullanılan çok yönlü bir biomaterial vardır peptid nanofibers5,6,7,8,9 sonrası derleme değişiklikler nispeten keşfedilmemiş bir alan kalır.
Dinamik disülfür exchange ve thiol Kimya supramolecular yapıları yüzeyde yeni ve işlevsel Biyomalzeme verim potansiyeline sahip bir alandır. 1,2-dithiolane moieties (genellikle bir türevi lipoic asit (la) veya asparagusic asit (aa)) birleşme, lipozom sistemleri10,11, blok kopolimerler12,13ve olarak bildirilmiştir çapa yüzeyler14,15düzenleme. Burada, biz sentezi ve karakterizasyonu, N-terminus bir 1,2-dithiolane fonksiyonel grup16ile değiştirilir Alzheimer Hastaligi ile ilişkili Aβ peptid nucleating çekirdeğinden elde edilen bir kendi kendine montaj peptid raporu, 17. Elde edilen supramolecular lifleri şimdi amiloid lifleri18supramolecular yüzeyde disülfür-Satım ve thiol reaktivite çalışmaya deneysel bir platform olarak hizmet vermektedir.
Protocol
Representative Results
Discussion
Bu makalede bir N-terminal 1,2-dithiolane değiştiren Self montaj peptid saflaştırılması ve karakterizasyonu elde edilen supramolecular yapıları ve hem sentezi ayrıntılarını anlatılır. Rapor burada 1,2-dithiolane peptid sentez 3-(acetylthio) -2 dithiolane habercisi üretmek için bir tek adımlı sentez de dahil olmak üzere avantajı vardır-(acetylthiomethyl) propanoic asit ve üzerinde reçine mikrodalga deprotection tepki Grup olarak toksik hidrazin deprotection için daha güvenli bir alternatif daha ö…
Declarações
The authors have nothing to disclose.
Acknowledgements
Yazarlar TEM Connecticut Devlet kolej ve üniversite (CSCU) Merkezi nanoteknoloji ve Dr Ishita Mukerji Wesleyan Üniversitesi’nde için erişim için onu CD kullanarak yardım ve Dr. B. Ellen Scanley onun teknik eğitim için teşekkür etmek istiyorum Spektrofotometre. Raporlanan çalışma kısmen Bilim Enstitüsü Üniversitesi Fairfield, NASA Connecticut alan Grant Konsorsiyumu tarafından ve Ulusal Bilim Vakfı Hibe numarası CHE-1624774 altında tarafından desteklenmiştir.
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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