方法文章

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

DOI：

10.3791/58135

⸱

2018年8月20日

 ,  , 
1Department of Chemistry and Biochemistry, Fairfield University

本文内容

摘要

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A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

摘要

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This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the Aβ peptide associated with Alzheimer's disease, the peptide is shown to self-assemble into cross-β amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

引言

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The robust peptide bond forming chemistry involved in solid-phase peptide synthesis and the ability to control sequence length and composition make the peptides that self-assemble into supramolecular structures a heavily researched field. The factors that control and stabilize peptide self-assembled structures, including side chain steric and electrostatic interactions, hydrogen bonding, and hydrophobic effects1, serve as a set of design rules. As the research into these fundamental design rules continues to progress, the logical next step in peptide self-assembly involves expanding the diversity of peptide-based structures and functions. While self-assembling peptides are a versatile biomaterial that have been used for many biomedical applications by tuning the peptide sequence or assembly conditions2,3,4, the development of strategies for post-assembly modifications to peptide nanofibers5,6,7,8,9 remains a relatively unexplored area.

Dynamic disulfide exchange and thiol chemistry at the surface of supramolecular structures is one area that has the potential to yield new and functional biomaterials. The incorporation of 1,2-dithiolane moieties (commonly a derivative of lipoic acid (la) or asparagusic acid (aa)) have been reported in liposome systems10,11, block copolymers12,13, and as organizing anchors at surfaces14,15. Herein, we report the synthesis and characterization of a self-assembling peptide derived from the nucleating core of the Aβ peptide associated with Alzheimer's disease that is modified at the N-terminus with a 1,2-dithiolane functional group16,17. The resulting supramolecular fibers now serve as an experimental platform to study the disulfide-exchange and thiol reactivity at the supramolecular surface of amyloid fibers18.

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方案

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1. Synthesis and Purification of 1,2-Dithiolane Modified Peptide

  1. Synthesis of dithiolane precursor, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid19.
    1. Add 1 g of 3-bromo-2-(bromomethyl)propionic acid (1 equiv.) dissolved in minimal amount of 1 M NaOH (approximately 4 mL) to a 25-mL round bottom reaction flask with stirring at 55 °C. Seal the reaction flask with a septa and place under nitrogen atmosphere.
    2. Prepare a solution containing 1.49 g of potassium thioacetate (3.2 equiv.) in 4 mL of deionized water and 3 mL of 2 M sulfuric acid (H2SO4) to create thioacetic acid in situ.
    3. Pull the thioacetic acid solution into a plastic disposable 10 mL syringe and place a needle on the syringe. Add the mixture dropwise to the reaction flask by piercing through the septa with the needle. Continue the reaction overnight at 55 °C.
    4. Monitor the reaction by thin layer chromatography (TLC) on silica gel 60 F254 plates using a mixture of methanol and dichloromethane (1:9). Visualize the reaction progress by bromocresol green stain. The product has an Rf = 0.57.
    5. After the reaction is complete and cooled to room temperature, acidify the mixture to pH 1 with 2 M H2SO4. A yellow oil separates out of solution.
    6. Extract the product with cold chloroform (40 mL x 3). Combine the organic layers and dry over magnesium sulfate. Remove the chloroform under reduced pressure.
    7. Confirm the identity of the isolated product, 1, by nuclear magnetic resonance (NMR) spectroscopy as shown in Figures 1B and C. Expect the following results: 1H NMR, CDCl3, 300 MHz: d = 10.1 (b, 1 H), 3.2 (m, 4 H), 2.9 (m, 1 H), 2.4 (s, 6 H); 13C NMR, CDCl3, 75 MHz: d = 195.1 (CH3COS-), 177.6 (-COOH), 45.1 (-CH2S-), 30.5 (CH), 29.2 (-SCOCH3).
      Note: The product is a yellow oil and has an overall yield of 83%. Use the product without further purification.
  2. SPPS and On-Resin Coupling of Dithiolane Precursor
    Note: The solid-phase peptide synthesis described below was conducted on an automated peptide synthesizer, following the recommended manufacturer’s protocols. Settings and reagents may be adapted for other commercial instruments or when using specialized amino acids.
    1. Weigh out 0.156 g of Rink amide 4-methylbenzhydrylamine (MBHA) resin (0.1 mmol) and place in a reaction vessel. Swell the resin in dimethylformamide (DMF) for at least 15 min prior to the start of the synthesis.
    2. Weigh out 4 equivalents of each fluorenylmethyloxycarbonyl (Fmoc) protected amino acid (0.4 mmol) in the sequence and 0.152 g of N,N,N’,N’-tetramethyl-O-(1H-benotriazol-1-yl)uranium hexafluorophosphate (0.4 mmol, HBTU) for each amino acid in the sequence. Each cartridge contains both the Fmoc-protected amino acid and HBTU.
    3. After running all pre-synthesis checks on the synthesizer (refill reagents, flushed reagent lines and pressurized all reagent bottles), place the amino acid cartridges into the carousel in the C’ to N’ terminus direction. Place an empty cartridge after the final amino acid position for the final N-terminal Fmoc deprotection step.
    4. Synthesize the peptide using the standard recommended settings.
      1. Deprotect the Fmoc group from the resin with 5 mL of 20% piperidine in DMF (5 min x 2).
      2. Wash the resin with DMF (6 x 5 mL) before the coupling step.
      3. For a single coupling step, add 4 mL of 0.4 M N-methylmorpholine in DMF to the Fmoc-protected amino acid and HBTU. Activate the Fmoc-protected amino acid solution for 30 s before transferring the solution to the reaction vessel.
        Note: The automated peptide synthesizer mixes the resin and the solution by bubbling N2 gas every 30 s for 20 min while the coupling reaction takes places. For manual peptide synthesis, place the reaction vessel on an orbital shaker at low speed for the duration of the coupling step.
      4. Drain the solution and wash the resin with DMF (3 x 5 mL).
      5. Repeat steps 1.2.4.1 through 1.2.4.4 for each Fmoc-protected amino acid in the C’ to N’-terminus direction to synthesize the peptide of interest.
    5. After the final N-terminal deprotection step, transfer the resin into a disposable fritted syringe. Wash the resin with DMF (3 x 5 mL) and dichloromethane (DCM, 3 x 5 mL).
      Note: The resin may be stored after DCM washing in a vacuum desiccator. If the resin was stored previous to coupling, ensure to swell the resin in DMF before the coupling reaction.
    6. Couple the dithiolane precursor (1) to the N-terminus of the on-resin peptide by adding 4 equivalents of 1, 5 mL of DMF, 4 equivalents of HBTU and 10 equivalents of N,N-diisopropylethylamine (DIPEA). Pre-activate the coupling mixture for 10 min before adding to the resin containing fritted syringe.
    7. Shake the coupling reaction for 2 h. After 2 h, wash the resin with DMF (3 x 5 mL) and repeat the coupling reaction with shaking overnight.
    8. After the second coupling, wash the resin with DMF (3 x 5 mL) and DCM (3 x 5 mL).
      Note: The resin can be stored at this point under vacuum until cleavage.
  3. Thioacetate Deprotection and Peptide Cleavage from Resin
    1. To deprotect the thioacetate group from the N-terminus dithiolane precursor, transfer the dried resin to a 10 mL microwave reaction tube and add 2 mL of DMF. Allow the resin to swell, add a small magnetic stir bar to the vessel, and re-suspend with a low speed of magnetic stirring for 15 min.
    2. Add 2 mL of concentrated ammonium hydroxide, cap the reaction vessel with the silicone septa, and place the reaction vessel into a microwave reactor using the microwave settings of 75 °C for 45 min with stirring.
    3. After the microwave reaction is complete, transfer the resin into a clean disposable fritted syringe. Wash with DMF (2 x 5 mL) and methanol (MeOH, 2 x 5 mL).
    4. Add a solution of concentrated ammonium hydroxide in methanol (1:4), for a total volume of 5 mL. Leave to shake overnight to increase intramolecular oxidation of disulfide bond in dithiolane ring.
    5. Wash the resin with MeOH (2 x 5 mL) and DCM (3 x 5 mL).
      Note: The dried resin may be stored in a vacuum desiccator at this point.
    6. Add the cleavage cocktail to the resin containing syringe with gently shaking for 1.5 h. The cleavage cocktail used is 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% water at a total volume of 5 mL.
      CAUTION: Work under chemical fume hood only. TFA is volatile and corrosive.
      Note: For the majority of peptide sequences and amino acid sidechain protecting groups, the above cleavage cocktail solution is sufficient; however, alternative cleavage cocktails may be needed for certain amino acid side chain protecting groups (in particular peptides containing Cys, Met, Trp and Arg) or other resin chemistry20.
    7. Precipitate the crude peptide into 25 mL of cold diethyl ether in a 50 mL conical tube by dropwise addition from the fritted syringe. The peptide precipitates as a white solid. Pellet the peptide by centrifugation at 1300 x g for 10 min. Carefully decant the diethyl ether into a separate container for waste collection.
    8. Add another 25 mL of diethyl ether to the conical tube and re-suspend the precipitate by vortexing. Repeat the centrifugation at 1300 x g for 10 min and decant the diethyl ether again. Dry the pellet under vacuum.
  4. Purification of 1,2-Dithiolane Modified Peptide
    Note: Purify the crude peptide by reverse phase-HPLC. Collect and combine the peptide peaks and confirm the mass by MALDI-TOF mass spectrometry.
    1. Dissolve the crude peptide pellet in minimal amount of acetonitrile with 0.1% TFA. Due to the peptide’s hydrophobicity and aggregation propensity, gently heat the sample at 40 °C to aid in solubility.
      Note: Avoid higher temperatures and sonication of the peptide in order to prevent potential disulfide exchange reactions21,22,23.
    2. To prepare 1 mL of crude peptide for HPLC purification, add 400 μL of concentrated peptide stock in acetonitrile to 600 μL of H2O with 0.1 % TFA and filter through a 22 μm syringe filter into a HPLC vial. An additional 5% isopropanol can be added to prevent peptide aggregation and precipitation.
    3. Purify the peptide using C-18 semi-preparative column with a flow rate of 3 mL/min over a linear gradient of 15-55% acetonitrile in 20 min. Set the UV detectors to 222 nm (amide backbone) and 330 nm (disulfide bond). Collect and combine the peaks of interest (Figure 2A).
    4. Confirm the peptide product mass by MALDI-TOF mass spectrometer in the reflectron mode (Figure 2B). For analysis, mix 0.5 μL of collected peak on the MALDI plate with 0.5 μL of 2,5-dihydroxybenzoic acid (DHB) matrix (10 mg/mL DHB in 50% acetonitrile, 0.1% TFA).
      Note: Common adducts in MALDI-TOF mass spectrometry include the sodium and potassium salt adduct ([M+Na]+ and [M+K]+) peaks. Desalting the sample prior to the analysis is recommended if the salt adduct peaks suppress the signal of the main [M+H]+ peak. Additionally, an oxidized peak of [M+O]+ is also detected in the 1,2-dithiolane modified peptide. A report on laser induced oxidation from the MALDI ionization using DHB matrix suggests that factors including sample concentration, solvent, and laser intensity can be modified to limit the MALDI induced oxidation artifact24.
    5. After MALDI-TOF confirmation of the correct mass, lyophilize the peptide after flash freezing. Keep the lyophilized peptide powder under vacuum until assembly.

2. Characterization of Supramolecular Self-Assembly Structures

  1. Formation of Amyloid Fibers
    1. To prepare self-assembly solution, weigh out 1 mg of the peptide powder using an analytical balance. Dissolve into a mixture (pH 7.5) of 20% acetonitrile and 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in a 1.5 mL microcentrifuge tube, to a final concentration of 1 mg/mL peptide assembly mixture. Vortex the assembly solution and leave to assemble at room temperature.
  2. Spectroscopic Characterization of Amyloid Fibers
    1. Follow the peptide assembly process via Fourier-transform infrared (FTIR) spectroscopy every few days. A broad peak centered around 1670 cm-1 is the IR signature arising from unassembled peptides in the sample17. The peptide assembly samples usually take one to two weeks for the broad unassembled peak to disappear and reach maturation.
      1. Dry an aliquot of 8-10 μL of the assembly solution as a thin film on the ATR diamond crystal. Monitor the disappearance of a large and broad water peak from 1640 to 1630 cm-1 as the dry film forms.
      2. Acquire IR spectra from 1500-1800 cm-1 averaging 50 scans with a 2 cm-1 resolution. Acquire and subtract the background scans prior to each sample scan. The IR signature for β-sheet assembly is a sharp peak between 1625 and 1635 cm-1 (Figure 3A)25,26.
    2. Characterize the peptide assembly into β-sheet rich supramolecular structures by circular dichroism (CD). Record the spectra using a CD spectropolarimeter with a Peltier temperature control system.
      1. Pipette 30 μL of the assembly solution in a 0.1 mm path length microcuvette.
        Note: A cell holder is needed to clamp and position the short path length cell in the instrument.
      2. For each spectrum, set the CD instrument to the following parameters: scanning wavelengths of 300 nm to 180 nm, scanning rate of 100/min, band width of 1 nm, 25 °C, average of three scans.
      3. Collect a spectrum of the buffer (20% acetonitrile/10 mM HEPES, pH 7.5) and subtract from each sample scan as a control. The CD signature for β-sheets is an ellipticity minimum centered around 220 nm (Figure 3B)27.
  3. Microscopy of Amyloid Fibers
    1. Allow two to three weeks for the peptide samples to mature into β-sheet rich supramolecular structures.
      Note: The assemblies can be imaged using transmission electron microscopy (TEM) at earlier stages of the assembly process as well.
      1. Pipette 10 μL of the peptide assembly solution onto the surface of TEM carbon grid.
        Note: Take care not to touch the pipette tip to the grid surface. High precision, self-closing tweezers are used to hold the TEM grid during preparation.
      2. Wait 1-2 min to allow the assemblies to adsorb onto the grid surface. Remove excess sample by touching filter paper to the edge of the grid.
      3. Prepare a 2% uranyl acetate stain by adding 100 μL of deionized water to commercially available 4% uranyl acetate solution. Pipette 10 μL of the 2% uranyl acetate stain onto the grid surface and incubate for 2-3 min. After the incubation, remove excess stain by touching filter paper to the edge of the grid.
      4. Place the TEM grids in a vacuum desiccator overnight. Store under vacuum until imaging.
      5. Image the prepared samples with TEM (Figure 3C). Typical parameters for microscopy are as follows: images at magnifications ranging from 9,300X to 23,000X, tungsten filament with an accelerating voltage of 120 kV.
        Note: ImageJ can be used to measure average fiber width of the supramolecular structures of acquired TEM images28.
        CAUTION: Please consult all relevant safety data sheets (SDS) before use. Several chemicals used in the synthesis, purification, and characterization of the described 1,2-dithiolane modified self-assembling peptides are corrosive or toxic and should only be used under a chemical fume hood. Always use appropriate personal protective equipment (including safety glasses, lab coat, full length pants, closed-toe shoes) when working in the laboratory.

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结果

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Aside from the initial one-step synthesis of the dithiolane precursor molecule, the rest of the 1,2-dithiolane modified peptide synthesis occurs on solid support (Figure 1A). The conversion of 3-bromo-2-(bromomethyl)propionic acid to 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, the dithiolane precursor, is confirmed by 1H and 13C NMR (Figure 1B and C) before it is coupled to the free N...

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讨论

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This article discusses the details of both the synthesis and the purification of an N-terminal 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting supramolecular structures. The synthesis of the 1,2-dithiolane peptide reported here has the advantages, including a one-step synthesis to produce the dithiolane precursor, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the on-resin microwave deprotection reaction of the precursor thioacetate protecting group to yield the oxidized ...

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披露

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The authors have nothing to disclose.

致谢

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The authors would like to thank Dr. B. Ellen Scanley for her technical training and help using the TEM at the Connecticut State Colleges and University (CSCU) Center for Nanotechnology and Dr. Ishita Mukerji at Wesleyan University for access to her CD spectrophotometer. The work reported was in part supported by the Science Institute at Fairfield University, the NASA Connecticut Space Grant Consortium, and by the National Science Foundation under Grant Number CHE-1624774.

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材料

本文使用的材料清单
姓名公司目录编号评论
溜冰酰胺 MBHA树脂，高负载Gyros Protein TechnologiesRAM-5-HL避免接触皮肤和眼睛;不要吸入
N，N-二甲基甲酰胺Fisher ScientificD119-4易燃液体和蒸气;刺激眼睛和皮肤;使用个人防护装备;远离明火
Fmoc-L-Val-OHGyros Protein TechnologiesFLA-25-V佩戴个人防护设备;不要吸入
Fmoc-L-Leu-OHGyros Protein TechnologiesFLA-25-L穿戴个人防护设备;不要吸入
Fmoc-L-Lys（Boc）-OHGyros Protein TechnologiesFLA-25-KBC佩戴个人防护设备;不要吸入
Fmoc-L-Phe-OHGyros Protein TechnologiesFLA-25-F穿戴个人防护装备;不要吸入
Fmoc-L-Ala-OHGyros Protein TechnologiesFLA-25-A穿戴个人防护装备;不要吸入
Fmoc-L-Gln（Trt）-OHGyros Protein TechnologiesFLA-25-QT穿戴个人防护装备;不要吸入
N，N，N′，N′-四甲基-O-（1H-苯并三唑-1-基）脲六氟磷酸Gyros Protein Technologies26432引起皮肤、眼睛和呼吸道刺激;不要吸入;在引擎下或通风良好的区域使用
DMF 中的0.4 M N-甲基吗啉Gyros Protein TechnologiesPS3-MM-L高度易燃;穿戴个人防护装备;远离热源，保持容器密闭;不要吸入或吞咽;在
DMF 中处理 20% 哌啶Gyros Protein TechnologiesPS3-PPR-L导致严重的眼睛和皮肤灼伤;易燃液体和蒸气;不要吸入
二氯甲烷Fisher ScientificD37-4可能导致癌症;不要吸入;穿戴个人防护装备;仅在引擎盖下使用;如果接触，遇水上升至少 15 分钟并就医
乙腈Fisher ScientificA998-4易燃;刺激眼睛;使用个人防护装备;只能在通风橱下使用;远离明火或热表面;如果接触到，用水冲洗至少15分钟，并就医
三氟乙酸Fisher ScientificA116-50引起严重烧伤;不要吸入;对水生生物有害;使用个人防护设备;只能在通风橱下使用;如果接触到，用水冲洗至少15分钟并立即就医
4%醋酸铀酰显微镜科学22400-4不要吸入;对水生生物有害
4-（2-羟乙基）哌嗪-1-乙磺酸Acros OrganicsAC172571000不要吸入;在室外或通风良好的地方使用
气 气体TechAir内容物在压力下，如果加热可能会爆炸
3-溴-2-（溴甲基）丙酸Alfa AesarAAA1963014不要吸入;对皮肤和眼睛造成刺激;腐蚀性
氢氧化钠Fisher ScientificS318-100使用个人防护设备;只能在通风橱下使用;如果接触，冲洗区域至少15分钟并获得医疗护理
代乙酸钾Acros OrganicsAC221300250引起皮肤和眼睛刺激;不要吸入;使用个人防护设备
硫酸Fisher ScientificSA213导致灼伤;远离水;远离可燃物;不要吸入;使用个人防护设备;如果接触冲洗区域至少 15 分钟并获得医疗护理
氯仿-dAcros OrganicsAC320690075可能的癌症危险;刺激皮肤和眼睛;不要吸入;使用个人防护装备;仅在通风橱下使用;如果接触冲洗区域至少 15 分钟并获得医疗护理
氯仿Fisher ScientificC298-4可能有癌症危险;刺激皮肤和眼睛;不要吸入;使用个人防护装备;仅在通风橱下使用;如果接触冲洗区域至少 15 分钟并获得医疗护理
N，N-二异丙基乙胺Acros OrganicsAC367841000高度易燃;对水生生物有害;佩戴个人防护设备;不要吞咽
氢氧化铵Fisher ScientificA669S-500腐蚀性物质;不要吸入
甲醇Fisher ScientificA452-4易燃液体和蒸气;使用个人防护设备;不要吸入;如果接触冲洗区域至少 15 分钟并就医
三异丙基硅烷Sigma Aldrich233781易燃;使用个人防护安全设备;保持容器密闭
二乙醚Fisher ScientificE138-1极易燃;对皮肤和眼睛有刺激性;使用个人防护设备
2,5-二羟基苯甲酸Sigma Aldrich39319-10x10MG-F不要吸入;刺激皮肤和眼睛
α-氰基-4-羟基肉桂酸阿尔法-伊撒AAJ67635EXK
c18 拉链尖端米利波尔ZTC18S096
三（2-羧乙基） 磷酸盐酸盐Thermo ScientificPI20490
硅胶 60 F254 涂层铝背 TLC 板EMD Millipore1.05549.0001
薄壁精密核磁共振管Bel-Art663000585外径 5mm
全塑料 Norm-Ject 注射器Air TiteAL10
一次性针头BD PrecisionGlideBD305185用过的针头被丢弃在锐器废物容器中
一次性烧结注射器TorviqSF1000LL10mL 烧结注射器在报告中被使用，但如果需要进行更大规模的合成，可以使用更大的注射器。
碳网格Ted Pella， Inc.CF200-CU确保在碳网格侧准备样品和染色，而不是网格
自闭合镊电子显微镜科学78318-3X非常锋利的尖端，长度：120 mm
0.1 mm 短程池Starna Cells， Inc.20/C-Q-0.1易碎
10 mL 容器盖CEM909210
10 mL 压力容器CEM908035
Aeris 半制备 HPLC 色谱柱Phenomenex00F-4632-N0150 x 10 mm
比色皿支架Starna Cells， Inc.CH-2049使用短光程细胞时需要
PS3 肽合成仪Gyros Protein Technologies
DiscoverSP 微波反应器CEM
离心机HERMLEZ 206 A使用固定的 6x50 mL 转子
HPLC岛津紫外检测器
核磁共振光谱仪Avance， Bruker300 MHz
MALDI-TOF 质谱仪Axima Confidence、岛津
冻干机Millrock TechnologyBT85A
傅里叶变换红外光谱仪Alpha Tensor、布鲁克
透射电子显微镜Tecnai Spirit、FEI与 Gatan Orius 光纤 CCD 数码相机一起使用。在 CSCU 纳米技术中心访问
圆二色光谱偏振仪J-810，JASCO与六节帕尔贴温度控制器一起使用。在卫斯理大学访问。
盐 后彻底清洗皮肤 电子氮硫子的闪亮铜侧

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