Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

Transcript

This protocol enables the information-directed assembly of oligomeric sequences by dynamic covalent interactions, truly mimicking the sequence-selective hybridization of nucleic acids but with increased interstrand bond strength. Dynamic covalent assemblies are typically restricted to simple architectures owing to their limited capacity for bond rearrangement and error correction. In contrast, by raising along the concentration of a Lewis acid to affect bond dissociation and subsequently catalyze bond rearrangement, this technique mitigates the prevalent kinetic trapping limitations of the self-assembly systems.

This method could be employed in a variety of fields that utilize covalent bond rearrangement reactions, from covalent organic frameworks with exceptionally low defect rates to biomaterial tissue interfaces that are able to adapt and accommodate continued remodeling of the tissue substrate. When performing this technique for the first time, individuals may find kinetically trapped assemblies even after prolonged annealing times. We advise first-time users to attempt hybridizations between oligomers bearing exclusively aldehyde and exclusively amine residues, simplifying the encoded information and thus hybridization.

To begin this procedure, weigh 0.125 grams of FMOC photolabile solid support resin and add it to a fritted automated synthesizer reaction vessel. Insert the vessel into the microwave portion of the synthesizer. Fill the main solvent bottle with DMF and the deprotection bottle with a solution of 20%4-methylpiperidine in DMF and empty the waste.

Next, prepare one molar solutions of bromoacetic acid and DIC in DMF with total volumes of 1.5 milliliters for each residue in sequence and 0.47 milliliters of acetic anhydride to 4.53 milliliters of DMF to make a five milliliter capping solution. Prepare 0.5 molar solutions of each primary amine to be used in NMP. The total volume of each solution should be 2.5 milliliters for each residue of the appropriate primary amine plus an additional 2.5 milliliters.

Add all of the solutions to the automated synthesizer manifold. Using an automated peptide synthesizer, swell the resin, cleave the FMOC group, and perform the displacement reaction as outlined in the text protocol. After this, salinize the walls of a fritted glass reaction vessel equipped with a three-way stopcock by filling it to the top with a solution of 5%dichlorodimethylsilane in DCE.

Let it sit for 30 minutes then drain the vessel and wash it with DCE and methanol. After the vessel is dry, transfer the resin to it. Wash the resin three times with DCM using five milliliters for each wash while bubbling with nitrogen gas through one arm and pulling vacuum with another.

Dry and store the resin and the attached oligopeptoid until deprotection and cleavage. When ready to continue, re-swell the resin if it has been stored for more than a day by bubbling it with five milliliters of DMF for 10 minutes. Drain the vessel and add a small magnetic stir bar and three milliliters of dry DCM to the glass peptide vessel.

Weigh 0.1 equivalents of the palladium catalyst and 25 equivalents of phenylsilane per alloc group. Use a clamp to position the reaction vessel at an angle above the stir plate such that the resin undergoes gentle agitation while remaining suspended in the solvent and cap the vessel to prevent the DCM from evaporating. After one hour, filter off the solution and wash the resin three times with DCM using five milliliters per wash.

Then, repeat the alloc deprotection once more. Next, rinse the resin sequentially with methanol and DCM two times and transfer the resin and magnetic stir bar to a 20 milliliter vial. Submerge the resin in DMF, stir, and cleave as outlined in the text protocol.

Then, use a syringe filter to separate liberated oligopeptoid from the resin and remove the solvent under vacuum. Reconstitute the peptoids in a 50-50 mixture of water and acetonitrile and purify with reversed-phase preparative HPLC. Combine the purified fractions, then freeze and lyophilize them to yield an off-white powder.

Analyze the powder with ESI and MALDI mass spectrometry. To evaluate purity, perform analytical HPLC of the purified oligopeptoids. First, prepare 10 millimolar stock solutions of each oligopeptoid sequence used for self-assembly and a 10 millimolar stock solution of scandium triflate in anhydrous acetonitrile.

Add 20 microliters of each peptoid stock solution to a three milliliter vial equipped with a magnetic stir bar. Add 1.5 equivalents of the scandium triflate solution per potential amine bond from the stock solution and add enough water and acetonitrile to form 200 microliters of a 2%water and acetonitrile solution. Stir gently at 70 degrees Celsius for two hours for acetyl deprotection of the aldehyde and dissociation of all strands.

After this, charge the vial with 200 microliters of chloroform and two milliliters of water. Shake the vial gently. Allow the mixture to stand for at least 15 minutes.

Upon complete phase separation, extract the organic layer with a microliter syringe. Transfer the organic layer to a new vial and stir at 70 degrees Celsius for oligomer annealing which typically takes six hours. To demonstrate the ability of information-encoded peptoids to undergo sequence-selective dynamic covalent self-assembly into molecular ladders, a representative strand is synthesized and hybridized with its complementary peptoid sequence.

The monomers NPAM and NPAL are employed as dynamic covalent reactant pairs with NEEE aiding solubility of final self-assembled products. Upon completion of the solid-phase submonomer synthesis, the alloc group is removed. Prior to and after deprotection, portions of the resin were cleaved under 405 nanometer light and characterized by electrospray ionization mass spectrometry.

The sequence is purified by prep HPLC, then lyophilized to achieve an off-white powder and the purity is confirmed with analytical HPLC. The oligopeptoid is subsequently hybridized with its complementary sequence to afford an in-registry ladder confirmed by MALDI-MS. When performing this procedure, remember to allow sufficient time for the layers completely separate until the aqueous fraction becomes transparent, at least 15 minutes to ensure sufficient catalyst extraction.

After completing this assembly procedure, mass spectrometry should be performed to determine the extent of hybridization. Additionally, inductively coupled plasma mass spectrometry or fluorine NMR can be used to assess the scandium concentration post-extraction. While this technique was recently developed, we anticipate that the biomimetic approach described in our work will be instrumental in directing the future design and information-directed assembly of complex nanostructures.

Several of the reagents used here are hazardous. Please use caution when handling these or any other chemicals. Wear all personal protective equipment and perform all experiments inside of a fume hood.

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Journal (Chemistry)

Journal (Chemistry)

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

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