1, 2-ジチオランの合成とキャラクタリゼーション自己組織化ペプチド
Summary
1, 2-ジチオランの合成のためのプロトコルは、ペプチドやペプチドの自己集合結果超分子構造の評価に変更されました。
Abstract
本報告では N 末端の 1, 2-ジチオランの合成に焦点を当てて自己組織化ペプチドとその結果の評価を変更された自己組織化超分子構造。合成経路は、ジチオラン前駆体分子 3-(acetylthio)-2 の樹脂の結合の固相ペプチド合成の活用-(acetylthiomethyl) プロパン酸、およびマイクロ波を用いた thioacetate ペプチドの deprotection1, 2-ジチオランに屈する前に、ガレージから最終的な胸の谷間 N 末端ペプチドを変更しました。アルツハイマー病に関連付けられている a β ペプチドの核のコアから派生した、1, 2-ジチオラン ペプチドの高速液体クロマトグラフィー (HPLC) 精製後、ペプチドはクロス β アミロイド線維に自己組み立てるに表示されます。フーリエ変換赤外分光法 (FT-IR)、円二色性分光法 (CD) と透過型電子顕微鏡 (TEM) によるアミロイド繊維を特徴付けるためのプロトコルが表示されます。組立後変更戦略を開発し、動的共有結合化学を探索するモデル系として、よ特徴付けられた自己組織化ペプチドを 1, 2-ジチオラン構造 N 末端修飾の方法を検討今することができます。超分子のペプチド ナノファイバーの表面。
Introduction
固相ペプチド合成に関与する堅牢なペプチド結合の化学を形成し、シーケンスの長さと構成を制御する機能、超分子構造に重く研究フィールドをまとめる自己ペプチド。制御し、側鎖の立体を含むペプチドの自己組織化構造と静電相互作用、水素結合、疎水性効果1、安定の要因はデザイン ルールのセットとして機能します。これらの基本的な設計ルールに研究が進み、ペプチドの論理の次の手順では自己ペプチド ベースの構造と機能の多様性を拡大して含まれます。自己組織化ペプチドがペプチド シーケンスまたはアセンブリ条件2,3,4のための戦略の開発をチューニングすることによって多くの医用に使用されている汎用性の高い生体材料ペプチド ナノファイバー5,6,7,8,9組立後変更のまま比較的未踏の領域です。
動的ジスルフィド交換とチオール化学超分子構造体の表面では、機能的な新しい生体材料をもたらす可能性がある 1 つの領域です。1, 2-ジチオラン鎖 (一般的リポ酸 (la) やアスパラガス酸 (aa) の派生物) の設立がリポソーム システム10、11ブロック共重合体12,13と報告されています。表面14,15でアンカーを整理します。ここで、合成と 1, 2-ジチオラン機能グループ16、N 末端修飾アルツハイマー病に関連付けられている a β ペプチドの核のコアから派生した自己組織化ペプチドの特性を報告します。 17。結果として得られる分子繊維は今アミロイド繊維18の超分子表面にチオール、ジスルフィド交換反応性を研究する実験的プラットフォームとして機能します。
Protocol
1. 1, 2-ジチオランの合成・精製ペプチドを変更 ジチオラン前駆体、3-(acetylthio)-2 の合成-(acetylthiomethyl) プロパン酸19。 3 の 1 グラムを追加-ブロモ – 2-(ブロモメチル) プロピオン酸 (1 当量) 最小限の 55 ° C で攪拌しながら 25 mL 丸底反応フラスコに 1 M 水酸化ナトリウム (約 4 mL) に溶解隔壁を有する反応フラスコを密封し、窒素雰囲気下で配置します。…
Representative Results
ジチオラン前駆体分子の初期段階合成、脇から 1, 2-ジチオラン変更ペプチド合成の残りの部分は、固体支持体 (図 1 a) で発生します。3 の変換-ブロモ – 2-(ブロモメチル) プロピオン酸 3-(acetylthio)-2- 1H (acetylthiomethyl) プロパン酸、ジチオラン前駆体を確認し、 13C の NMR (図 1 bとC) それの前に自由に?…
Discussion
この記事では、両方の合成の詳細は、N ターミナル 1, 2-ジチオラン修正の自己組織化ペプチドの精製と結果の超分子構造の性質をについて説明します。ここで報告される 1, 2-ジチオラン ペプチドの合成ジチオラン前駆体、3-(acetylthio)-2 を生成するワンステップ合成を含む利点があります-(acetylthiomethyl) プロパン酸と樹脂の電子レンジの脱保護反応、有毒なヒドラジンの脱保護する安全な代替案…
Disclosures
The authors have nothing to disclose.
Acknowledgements
著者博士 B. エレン Scanley をありがとう彼女の技術的なトレーニングと彼女の CD にアクセスするためのナノテク ・ ウェスリアン大学で博士中川ムカージについてコネチカット州立大学および大学 (CSCU) センターで TEM を使用してヘルプをしたいです。分光光度計。報告される作業は、フェア フィールド大学、NASA コネチカット宇宙グラント コンソーシアムの科学研究所、国立科学財団助成番号チェ-1624774 の下に一部サポートされていました。
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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Cite This Article
Neves, R., Stephens, K., Smith-Carpenter, J. E. Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides. J. Vis. Exp. (138), e58135, doi:10.3791/58135 (2018).