点赞

收藏

分享

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

互联网2013-11-13

2161

实验材料

Name	Company	Catalog Number	Comments
Dimethylformamide	EMD	EM-DX1726P-1	99 %
N-methylpyrrolidinone	BDH	BDH1141-4LP	99%
Bromoacetic Acid	Acros Organics	200000-106	99%
4-Methylpiperidine	Sigma Aldrich	M73206	96%
N,N’-diisopropylcarbodiimide	Chem-Impex	001100	99.5%
Dichloromethane	EMD	EMD-DX0835	ACS grade
Acetonitrile	EMD	EM-AX0145P-1	99.8%
Trifluoroacetic acid	Sigma Aldrich	T6508	99%
Triisopropylsilane	Sigma Aldrich	233781-10G	For TFA cleavage
1,2-Dichloroethane	JT Baker	JTH076-33	For siliconization of glass reaction vessels
Phenethylamine	Sigma Aldrich	407267-100ML	>99.5% Hydrophobic side-chain amine
Boc-ethylenediamine	CNH Technologies	C-1112	Cationic side-chain amine
t-Butyl beta-alanine HCl	Chem-Impex International	04407	Anionic side-chain amine
α-Cyano-4-hydroxycinnamic acid	Sigma Aldrich	C8982-10X10MG	For MALDI matrix
Nile Red	Sigma Aldrich	19123-10MG	For fluorescence Imaging
Dichlorodimethylsilane	Sigma Aldrich	80430-500G-F	For siliconization of glass reaction vessels
Disposable PP fritted cartridge	Applied Separations	2416	6 mL polypropylene cartridge with 20 mm PE frit
Disposable 3 way luer adapter	Cole Parmer	31200-80	Stopcock for disposable manual synthesis reaction vessel
Luer Lock ring	Cole Parmer	45503-19	?” fitting for disposable manual synthesis reaction vessel
Fittings Luer	Cole Parmer	45500-20	?” fitting for disposable manual synthesis reaction vessel
Disposable PP pipets	VWR	16001-194	For TFA transfers
Luer lock plastic syringe	National Scientific	S7515-5	6 mL syringes
1 dram glass vial	VWR	66011-041	With phenolic molded screw cap with polyvinyl-faced pulp liner
20 mL scintillation vial	VWR	66022-060	With attached PP cap and pulp foil liner
Secure-Seal adhesive spacer	Invitrogen	S-24736	For fluorescence imaging
Glass slides	Electron Microscopy Sciences	63411	For fluorescence imaging
Cover slip	VWR	48366-067	For fluorescence imaging
4” Silicon wafer	Ted Pella	16007	Pre-dice in 5x7 mm chips
0.45 filter	VWR, Acrodisc	28143-924	For HPLC. PTFE membrane
Agarose	BD	212272	For fluorescence imaging
SPE Vacuum Manifold	Sigma Aldrich	57044	Example of SPE vacuum manifold
Fritted glass vessel	Ace glass	6402-12	Porosity C frit
Plasma Cleaner/Sterilizer	Harrick Plasma	PDC-32G	Example of plasma cleaner to prepare silicon chips for SEM

实验步骤

1. Solid-Phase Submonomer Synthesis of Polypeptoids

Solid-phase synthesis (SPS) is a common technique used to synthesize sequence-specific biopolymers step-wise, directly on an inert solid-support such as a polymeric resin bead. High coupling yields and ease of excess reactant removal are major advantages of SPS. After a coupling reaction to the resin, excess reagents are simply drained and the beads are washed to be ready for the next reaction step. After the final synthesis reaction, the full-length oligomers are cleaved from the resin and the solution-phase material can be further studied. Here, we adapt the SPS procedure to generate sequence-specific peptoid polymers.

1) Setup: All steps of the manual peptoid synthesis can be carried out in a disposable, polypropylene (PP) fritted cartridge or a fritted glass reaction vessel equipped with a 3-way stopcock. Perform all operations in a fume hood. For incubations in the glass vessel or plastic cartridge, connect one arm to a nitrogen supply to gently bubble the solution for proper mixing. Alternatively, for reaction incubations in the disposable cartridge, seal both ends of the cartridge with caps and place on a rotary shaker. To drain reaction mixtures or washes, connect to house vacuum via a waste trap. The vessel should be fritted with a coarse frit. Siliconize the glass reaction vessel to avoid beads from sticking to the walls of the glass vessel. Prepare a solution of 5% dichlorodimethylsilane in dichloroethane (v/v). Fill a clean and dry reaction vessel to the top with siliconizing solution, let sit for 30 minutes, then drain. Wash the vessel once with DCE and then once with methanol. The siliconizing solution can be reused, so it should be saved. Either air dry or shake off any excess solution and bake the glassware until dry after removing the stopcock. Cool reaction vessel before adding resin.

2) Add 100 mg (0.06 mmol) of Rink amide resin to a fritted reaction vessel. Swell the resin by adding 2 mL of dimethylformamide (DMF). Agitate by shaking or bubbling for 10 minutes. Drain the solution by vacuum to isolate the swelled resin.

3) Add 1 mL of 20% 4-methylpiperidine in DMF (v/v) to deprotect the Fmoc group. Agitate for 2 minutes and drain. Repeat with a 12 minute incubation.

4) Rinse the resin by adding 2 mL of DMF, agitating for 15 seconds, and draining. Repeat 3x.

5) Bromoacetylation: Add 1 mL of 0.6 M bromoacetic acid (0.6 mmol) in DMF and 86 μL of N,N'-diisopropylcarbodiimide (0.93 equivalent, 0.56 mmol). Incubate with gentle bubbling for 30 minutes, then drain and rinse with 2 mL of DMF (repeat 4x).

6) Displacement: Add 1 mL of 1-2 M amine in N-methylpyrrolidinone. Incubate with bubbling for 30-120 minutes, then drain and rinse with DMF (4x 2 mL).

7) Continue to grow the peptoid chain by repeating the submonomer cycle, steps 1.5 (bromoacetylation) and 1.6 (displacement).

8) After the final displacement is done, rinse with 2 mL of DMF (repeat 4x), then 2 mL of dichloromethane (repeat 3x). Cap and store the reaction vessel until cleavage.

9) Pause in synthesis (optional): To pause during a peptoid synthesis, finish the displacement reaction and continue to step 1.8. To continue growing the peptoid chain, restart the synthesis by re-swelling the dried resin (step 1.2) and repeating the submonomer cycle (step 1.5 and 1.6). The resin can be dried and stored after any displacement except the 2nd displacement because the resin-peptoid conjugate may form a cyclic diketopiperazine side product.

10) For multiple simultaneous syntheses, a solid-phase extraction vacuum manifold is recommended to maximize efficiency. Peptoid synthesis can also be automated by properly programming methods in commercially available peptide synthesizers, such as the Aapptec Apex 396, CEM Liberty microwave synthesizer and Protein Technologies Inc. Prelude synthesizer.

2. Cleavage and Side-Chain Deprotection

1) Transfer all of dried resin to a 20 mL scintillation glass vial.

2) Working inside a hood and using proper personal protective equipment, add 4 mL of trifluoroacetic acid (TFA) cleavage cocktail¹ (e.g. 95% aq. TFA, see discussion) to the scintillation glass vial and cap tightly. Shake for 10 minutes to 2 hours at room temperature (see discussion).

3) Collect the TFA cleavage solution by filtering the resin through a disposable, PP fritted cartridge into a new, pre-weighed 20 mL scintillation glass vial. A disposable, PP pipette is convenient to transfer the cleavage cocktail solutions.

4) Add 1 mL of fresh cleavage cocktail to rinse the resin and collect any residual peptoid. Repeat 2x.

5) Evaporate TFA by blowing a gentle stream of nitrogen or by using a Biotage V10 evaporator.

6) Redissolve the crude oil in 6 mL of acetonitrile/water 1:1 (v/v) for HPLC. Freeze and lyophilize. Repeat.

7) Record the weight of the crude product. Store as a dry powder at -20 °C

8) Test cleavage (optional): A test cleavage on 0.5% of the resin can be performed to quickly determine the purity and mass of the synthesized peptoid and whether correct cleavage conditions were chosen. Test cleavages are especially useful to monitor the progress of the synthesis.

3. Characterization and Purification of the Polypeptoid

1) Through a combination of analytical HPLC, electrospray LC-MS, and/or MALDI-TOF, determine the purity of the crude product and whether the desired molecular weight is present.

2) Prepare a ˜5-10 mg/mL solution of the dry peptoid powder in water with minimal acetonitrile as needed for solubility. Filter clear solution of crude peptoid product with 0.45 μm syringe filter to remove dust and particles.

3) Analytical HPLC and electrospray LC-MS: Prepare a _˜ 20 μg/mL crude peptoid solution. Filter 200 μL with a 0.45 μm filter and inject 20 μL.

4) MALDI: Mix 1 μL of _˜ 20 mg/mL peptoid with 1 μL matrix. Spot 1 μL on MALDI plate and allow to air dry. Matrix and acquisition mode is dependent on sample (Fig. 5).

5) Purify the crude peptoid mixture with reverse-phase prep HPLC. Choose the gradient and column (C4 or C18) based on the hydrophobicity of polypeptoid. Combine purified fractions, freeze, and lyophilize, resulting in a fluffy white powder. Record the weight of the final product.

6) Formation of HCl salt (optional): Redissolve the lyophilized powder in 100 mM HCl (aq.) with minimal acetonitrile. Transfer to pre-weighed glass vial. Freeze and re-lyophilize. Repeat 2x. Reweigh to determine mass of peptoid powder.

4. Peptoid Nanosheet Formation

This section describes the protocol to form sheets from a single-chain, sequence specific, amphiphilic 36-mer peptoid (Fig. 1). After the peptoid strand is synthesized, purified, and lyophilized as described above, the resulting white powder is dissolved in DMSO to make a 2 mM stock solution.

1) Prepare 500 μL of 20 μM peptoid solution in sheet formation buffer (10 mM Tris-HCl, 100 mM NaCl, pH 8.0 in water) in a 1 dram glass vial. First, add 445 μL of Milli-Q water, 50 μL of 10x sheet formation buffer, and vortex to mix. Then, add 5 μL of 2 mM peptoid stock solution and gently swirl solution. Cap the glass vial.

2) Sheets are formed by the gentle agitation of the dilute aqueous peptoid solution. Slowly tilting the glass vial from the horizontal position to the upright position results in sheets. Gentle shaking also yields sheets; however, the sheets tend to be smaller and with fewer straight edges. A more thorough analysis of the sheet formation mechanism is reported separately.²

3) For many high-quality sheets, rotate the glass vials about the horizontal axis slowly (<1 RPM) for one to three days. An Appropriate Technical Resources RKVSD Rotamix tube rotator or a customized rocker can perform this continuously.

4) Dialysis of Nanosheets (optional): In certain applications, it may be necessary to remove any free peptoid chains or buffers/salts. Soak a Float-a-Lyzer 100 kD membrane in the desired buffer for 15 minutes. Load 500 μL peptoid sheet solution into the sample chamber. Soak in 500 mL of desired buffer, stirring with a magnetic stir bar at 60 rpm. Allow dialysis of sheets to proceed for 4 hours. Every hour, exchange with a fresh stock of buffer solution.

5. Fluorescence Microscopy of Nanosheets

1) Fluorescence images of the nanosheets were imaged with Nile Red, an environmentally sensitive dye whose fluorescence intensity increases when it is localized in hydrophobic environments (Fig. 2).

2) Add 1 μL of 100 μM Nile Red to 100 μL of the nanosheet solution to obtain a final concentration of 1 μM of Nile Red.

3) Make a 1% agarose solution in hot water and pour into a plastic Petri dish. Ensure the agarose solution is approximately 1/8 inch thick and allow the solution to cool undisturbed on a flat surface. After the agarose sets, use a spatula to cut and transfer 1 cm x 1 cm squares to a glass slide.

4) To collect the sheets in the same plane, spot 1 μL of sheet solution on the piece of agarose. Allow the agarose to absorb the buffer for 2 minutes, leaving the sheets at the surface. Image within 15 minutes, otherwise the agarose will begin to deform due to dehydration.

5) To image sheets in solution, load 15 μL inside a 20 mm diameter 0.12 mm gasket on a glass slide. Cover with a coverslip. If the sheets are simply sandwiched between a glass slide and coverslip without a gasket, many sheets will shear and the seemingly minor evaporation will cause the sheets to constantly move.

6) Image sheets under epifluorescence illumination (e.g. an Olympus IX81 inverted microscope fitted with an Andor iXonEM EMCCD spectra with a Texas red filter).

6. Scanning Electron Microscopy (SEM) of Nanosheets

1) Plasma etch of silicone substrate (optional): The silicon chips are plasma etched to aid in the adsorption of sheets. Place the silicon chips in the vacuum chamber of a plasma cleaner (e.g. Harrick Plasma Cleaner/Sterilizer PDC-32G). Pump down to 200 mTorr and set the RF coil to 18W (high setting for PDC-32G). Etch for 2 minutes.

2) Drop 20 μL of peptoid sheet solution on a plasma-treated silicon substrate. Allow to sit for 3 minutes. Remove excess solution with tip of Kim-wipe. Pipette 20 μL of water onto the surface and remove excess solution again to remove buffer and salts. Repeat 4x.

3) Alternatively, dialyze the peptoid sheet solutions against water to remove buffer and salt. Drop 20 μL of dialyzed sheet solution on plasma-treated silicon substrates. Air dry the sample.

4) Image sheets with SEM (e.g. Zeiss Gemini Ultra-55 Analytical Scanning Electron Microscope) with an in-lens detector and at beam energies between 1 kV and 5 kV (Fig. 3).

7. Safety Notes:

1) Dimethylformamide and Dichloromethane are reasonably suspected carcinogens.

2) N,N'-Diisopropylcarbodiimide, 4-methylpiperdine and bromoacetic acid are hazardous to the skin, eyes, and respiratory tract. They should be used in the hood with care. It may be toxic if inhaled or absorbed through the skin, and exposure may result in sensitization. Empty containers retain product residue (liquid/vapor) and should be thoroughly rinsed before removing them from the hood.

3) TFA is a strong acid, and is extremely destructive to the upper respiratory tract, eyes, and skin. TFA is also volatile-keep concentrated solutions of TFA in the hood at all times to avoid respiratory damage. Use proper PPE, and caution when handling solutions of TFA. Change gloves promptly if they come in contact with TFA, and immediately clean up any spills.

8. Representative Results:

This section describes the synthesis, characterization, and purification of a sequence-specific 36-mer peptoid chain that folds into a highly ordered nanosheet³ (Fig. 1).

The block-charge peptoid H-[Nae-Npe]₉ -[Nce-Npe]₉ -NH₂ was synthesized on 100 mg of Rink amide resin. A 2 M amine solution was used for all displacement reactions, which were carried out for 60 minutes for residue 1-18 and 120 minutes for residue 19-36. t-Butyl beta-alanine HCl was converted to the free base (see discussion) whereas phenethylamine and boc-ethylenediamine were both used directly. The resin was cleaved with 95% TFA, 2.5% triispropylsilane, 2.5% water for 2 hours. TFA was evaporated and the resulting viscous oil (~180 mg) was re-dissolved in 6 mL acetonitrile:water 1:1 (v/v). Product purity (Fig. 4) and presence of the product mass was confirmed by from analytical RP-HPLC (30-80% acetonitrile in water gradient, both containing 0.1% (v/v) TFA, at 1 mL/min over 30 minutes at 60 °C with a C18, 5 μm, 50 X 2 mm column) and MALDI (Fig. 5).

Purification with reverse phase HPLC on a Vydac C18 column (10 μm, 22 mm x 250 mm) proceeded, using a gradient of 30-60% acetonitrile in water with 0.1% TFA over 60 minutes at 10 mL/min. The column was loaded with 60 mg of crude product for each chromatographic run. The purified fractions were combined based on purity from analytical RP-HPLC (Fig. 4) and lyophilized to yield _˜ 80 mg of a fluffy white powder.

Purified block-charge peptoid molecular weight was confirmed by MALDI. 1 μL of 100 μM purified peptoid in acetonitrile:water 1:1 (v/v) was mixed with 1 μL of matrix (5 mg/mL α-cyano-4-hydroxycinnamic acid in acetonitrile:water 1:1 v/v and 0.1% TFA) and 1 μL was spotted on the MALDI plate. After the sample air-dried, it was placed in the Applied Biosystem/MDS SCIEX 4800 MALDI TOF/TOF Analyzer. The acquisition and processing modes were linear low mass. The calculated weight was entered in the targeted mass to automatically adjust for the delay time. The laser intensity was set to 3400. The observed mass, 4981.2, matches closely to the calculated mass of 4981.74.

The lyophilized purified powder was dissolved in DMSO to make a 2 mM stock solution, which can be stored at 4 °C. Sheets were prepared by aforementioned protocol and imaged with fluorescence optical microscopy and SEM (Fig. 2 and 3). A variety of shapes with feature sizes ranging up to 300 μm are observed, and notably, straight edges are prominent.

1. King, D.S., Fields, C.G., & Fields, G.B. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int. J. Pept. Pro. Res. 36, 255-266 (1990).

2. Sanii B., Kudirka, R., Cho, A., Venkateswaran, N., Oliver, G.K., Olson, A.M., Tran, H., Harada, R.M., Tan, L., & Zuckermann, R.N. Shaken, not stirred: Collapsing a peptoid monolayer to produce free-floating, stable nanosheets. J. Am. Chem. Soc. , DOI: 10.1021/ja206199 (2011).

3. Kudirka, R., Tran, H., Sanii, B., Nam, K.T., Choi, P.H., Venkateswaran, N., Chen, R., Whitelam, S., & Zuckermann, R.N. Folding of a single-chain, information-rich polypeptoid sequence into a highly-ordered nanosheet. Bioploymers: Peptide Science. 96, 586-595 (2011).

4. Utku, Y., Rohatgi, A., Yoo, B., Zuckermann, R., Pohl, N., & Kirshenbaum, K. Rapid multistep synthesis of a bioactive peptidomimetic oligomer for the undergraduate laboratory. J. Chem. Ed. . 87, 637-639 (2010).

5. Fowler, S.A. & Blackwell, H.E. Structure-function relationships in peptoids: Recent advances toward deciphering the structural requirements for biological function. Org. Biomol. Chem. 7, 1508-1524 (2009).

6. Zuckermann, R.N. & Kodadek, T. Peptoids as potential therapeutics. Curr. Op. Mol. Ther. 11, 299-307 (2009).

7. Chongsiriwatana, N.P., Patch, J.A., Czyzewski, A.M., Dohm, M.T., Ivankin, A., Gidalevitz, D., Zuckermann, R.N., & Barron, A.E. Peptoids that mimic the structure, function and mechanism of helical antimicrobial peptides. Proc. Natl. Acad. Sci. U. S. A. 105, 2794-2799 (2008).

8. Yam, A.Y., Wang, X., Gao, C., Connolly, M.D., Zuckermann, R.N., Bleua, T., Halla, J., Fedynyshyn, J., Allauzen, S., Peretz, D., & Salisbury, C.M. A Universal method for detection of amyloidogenic misfolded proteins. Biochem. 50, 4322-4329 (2011).

9. Huang, C.-Y., Uno, T., Murphy, J.E., Lee, S., Hamer, J.D., Escobedo, J.A., Cohen, F.E., Radhakrishnan, R., Dwarki, V., & Zuckermann, R.N. Lipitoids - novel cationic lipids for cellular delivery of plasmid DNA in vitro . Chem. Biol. 5, 345-354 (1998).

10. Schroeder, T., Niemeier, N., Afonin, S., Ulrich, A.S., Krug, H.F., & Bräse, S. Peptoidic amino- and guanidinium-carrier systems: Targeted drug delivery into the cell cytosol or the nucleus. J. Med. Chem. 51, 376-379 (2008)

11. Lee, B.-C., Chu, T.K., Dill, K.A., & Zuckermann, R.N. Biomimetic nanostructures: Creating a high-affinity zinc-binding site in a folded nonbiological polymer. J. Am. Chem. Soc. 130, 8847-8855 (2008).

12. Murnen, H.K., Rosales, A.M., Jaworski, J.N., Segalman, R.A., & Zuckermann, R.N. Hierarchical self-assembly of a biomimetic diblock copolypeptoid into homochiral super helices. J. Am. Chem. Soc. 132, 16112-16119 (2010).

13. Nam, K.T., Shelby, S.A., Marciel, A.B., Choi, P.H., Chen, R., Tan, L., Chu, T.K., Mesch, R.A., Lee, B.-C., Connolly, M.D., Kisielowski, C., & Zuckermann, R.N. Free-floating ultra-thin two-dimensional crystals from sequence-specific peptoid polymers. Nature. Mater. 9, 454-460 (2010).

14. Burkoth, T.S., Beausoleil, E., Kaur, S., Tang, D., Cohen, F.E., & Zuckermann, R.N. Toward the synthesis of artificial proteins: The Discovery of an amphiphilic helical peptoid assembly. Chemistry & Biology 9, 647-654 (2002).

15. Murphy, J.E., Uno, T., Hamer, J.D., Cohen, F.E., Dwarki, V., & Zuckermann, R.N. A Combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery. Proc. Natl. Acad. Sci. U. S. A. 95, 1517-1522 (1998).

16. Mora, P., Masip, I., Cortés, N., Marquina. R., Merino, R., Merino, J., Carbonell, T., Mingarro, I., Messeguer, A., & Pérez-Payá, E. Identification from a positional scanning peptoid library of in vivo active compounds that neutralize bacterial endotoxins. J. Med. Chem. 48, 1265-1268 (2005).

17. Zuckermann, R.N., et al. Discovery of nanomolar ligands for 7-transmembrane G-protein coupled receptors from a diverse (N-substituted)glycine peptoid Library. J. Med. Chem. 37, 2678-2685 (1994).

18. Alluri, P., Liu, B., Yu, P., Xiao, X. & Kodadek, T. Isolation and characterization of coactivator-binding peptoids from a combinatorial library. Moleular Biosystems. 2, 568-579 (2006).

19. Figliozzi, G.M., Goldsmith, R., Ng, S., Banville, S.C., & Zuckermann, R.N. Synthesis of N-(substituted)glycine peptoid libraries. Methods Enzymol. 267, 437-447 (1996).

20. Culf, A.S. & Ouellette, R.J. Solid-phase synthesis of N-substituted glycine oligomers (α peptoids) and derivatives. Molecules. 15, 5282-5335 (2010).

21. Burkoth, T.S., Fafarman, A.T., Charych, D.H., Connolly, M.D., & Zuckermann, R.N. Incorporation of unprotected heterocyclic side chains into peptoid oligomers via solid-phase submonomer synthesis. J. Am. Chem. Soc. 125, 8841-8845 (2003).

ad image

ad image

相关产品推荐

Recombinant-Cyanidioschyzon-merolae-Photosystem-I-assembly-protein-Ycf4ycf4Photosystem I assembly protein Ycf4

￥10234

过氧化物酶来源于辣根，9003-99-0，Highly stabilized, essentially salt-free, lyophilized powder, 200-300 units/mg solid (using pyrogallol)，阿拉丁

￥1030.90

HiFiScript gDNA Removal cDNA Synthesis Kit，阿拉丁

￥1047.90

纳米氧化铝，1344-28-1，≥99.9% metals basis, powder,α-phase,150nm，阿拉丁

￥102.90

过氧化物酶来源于辣根，9003-99-0，Type XII, essentially salt-free, lyophilized powder,≥250 units/mg solid (using pyrogallol)，阿拉丁

￥743.90

相关问答

问

请问大家有没有遇到过Gibson assembly 乱连的情况啊？

4 回答 680 围观

推荐阅读

Solid-Phase Synthesis of Phosphopeptides

DNA Origami: Synthesis and Self‐Assembly

Solid-Phase Supports for Oligonucleotide Synthesis

关于丁香通

公司信息

个人用户

企业机构

无忧采购轻松科研

无忧采购轻松科研

提问

扫一扫

丁香实验小程序二维码

实验小助手

丁香实验公众号二维码

扫码领资料

反馈

TOP

打开小程序