Synthesis of long peptoid molecules for nanomaterial assembly on Biotage® Initiator+ Alstra peptide synthesizer

Introduction

Peptoids are N-substituted glycine molecules that mimic peptide structures and offer a wide range of functionalities for drug discovery. First discovered by Chiron Corporation in 1992 (1), peptoids have since gained significant attention in fields ranging from 3D nanomaterial assembly (2,3) to bioactive molecules assisting antimicrobial (4–6) drug discovery and antiviral (7) agents.

Peptoid synthesis can be achieved via a monomer or sub-monomer approach, with the latter being more widely adopted in research laboratories (8–10). To explore peptoid-based nanomaterial assembly, we designed and synthesized a 14-mer peptoid and an N-lipidated 10-mer peptoid using the sub-monomer approach on solid support with the automated Biotage® Initiator+ Alstra™ microwave peptide synthesizer. We synthesized structurally diverse 14-mer and 10-mer peptoids (Figure 1) with 78% and 72% crude purities respectively, demonstrating an effective synthesis that allows us to adapt similar protocols for the synthesis of even longer peptoid chains.

Figure 1. Chemical structures of peptoids (Nae-Npe)7, 14-mer (A.) and GN2-Npm9-C8, 10-mer (B.) synthesized in this study.

Experimental

Reagents and materials

The reagents used were: trifluoroacetic acid (TFA), triisopropylsilane (TIPS), N,N-dimethylformamide (DMF), dichloromethane (DCM), acetonitrile (ACN), 4-methylpiperidine, formic acid, N,N-diisopropylcarbodiimide (DIC), N-methyl- 2-pyrrolidone (NMP), and bromoacetic acid (BrAcOH). The solvents and reagents were purchased from commercial sources. TentaGel SRAM (90 µm, 0.23 mmol/g loading capacity) was purchased from Iris Biotech GmbH. The amine building blocks: tert-Butyl N-(2-aminoethyl)carbamate (Nae), 2-phenylethylamine (Npe), tryptamine (Ntrp), benzylamine (Npm) and octylamine (Noc), BrAcOH, DIC, and TFA were purchased from Sigma-Aldrich. Amine building block N-Boc-1,4- butanediamine (Nlys) was purchased from BLDpharm. DMF, ACN and NMP were purchased from VWR BDH Prolabo or ChemSolute.
 

Synthesis and analysis

The synthesis of peptoids (Nae-Npe)7 and GN2-Npm9-C8 (Fig. 1) were performed on the automated Biotage® Initiator+ Alstra™ microwave peptide synthesizer, in a 10 mL reactor vial, by initially deprotecting 400 mg - for (Nae-Npe)7, respectively 300 mg - for GN2-Npm9-C8 of TentaGel™ S RAM resin (loading capacity of 0.23 mmol/g), for each peptoid with 4 mL of 20% piperidine in DMF, for 20 minutes, at room temperature. This same step was repeated to ensure complete deprotection before starting the sub-monomer cycle.
Bromoacylation and displacement steps were easily programmed using the system’s touchscreen interface as follows: For the bromoacylation step, 3 mL of bromoacetic acid (BrAcOH 0.6 M in DMF) were mixed with 270 µL DIC (1M in THF), for 15 seconds (total premixing operational time of 1 minute), followed by transfer of the mixture to the reaction vessel and reacted for 30 minutes at 40 °C. After the reaction, the resin was washed 4 times with 2 mL DMF. For the displacement step, 3 mL of the respective amine building block (1.0 M in NMP) was added to the reaction vessel and left to react for 1 hour at 40 °C.

After the displacement step the resin was washed 5 times with 3 mL DMF. Both steps were repeated until the desired chain length was synthesized. Registering each detail of the amines (details in supporting information) used for displacement (full name, one and three letter code, CAS, reagent molecular weight, and residue molecular weight) allowed the easy input of the sequence to be synthesized and full usage of the software’s auxiliary tools such as the product MW calculation, product mass calculation, and the reagent calculation table. For each residue of the peptoid sequence, one bromoacylation and one displacement step were added to the method, making up a full sub-monomer cycle. Following the last displacement step, the resin was washed 4 times with 3 mL DCM and the resin was allowed to dry, typically overnight in a desiccator.

Cleavage of the peptoid product from the resin was performed by treatment with a cleavage cocktail containing TFA:H2O:TIPS (96:2:2, v/v/v) in 2 steps using 3 mL of the cocktail in each step, where treatment for 30 min in the first step followed by 1 hour for the second cleavage treatment step. The reaction was carried out at room temperature, while shaking. After the peptoid was cleaved, the cleavage cocktail was evaporated by Biotage® V-10 Touch evaporation system, and the peptoid solubilized in H2O:ACN (70:30, v/v) mixture and lyophilized.

Analysis of the crude product was performed on Dionex Ultimate 3000 UHPLC and a diode array detector interfaced with a HESII electro spray ion source to a Thermo Finnigan LTQ-XL linear ion-trap mass spectrometer equipped with a Kinetex C18 LC column (2.6 μm, 100 × 2.1 mm) and wavelength UV detection (200-800 nm) and MS detection. The following solvent system was used: solvent A, H2O:ACN (90:10, v/v) containing 0.1% formic acid; solvent B, acetonitrile containing 0.1% formic acid. The column was eluted using a linear gradient from 5% – 100% of solvent B with a flow rate of 0.4 mL/min over 12 min.
 
Scheme 1. Schematic illustration of solid phase sub-monomer peptoid synthesis procedure. Elongation along the peptoid backbone occurs via a 2-step procedure, 1) bromo acetylation and 2) displacement step where the functionality is introduced via amine addition.

Results and discussion

Both peptoids sequences were assembled using sub-monomer syntheses as described above with microwave heating during the coupling steps (Scheme 1). The 14-mer, (Nae-Npe)7 crude peptoid was obtained with a purity of 78 % and its identity confirmed according to the average isotopic composition for C98H136N22O14 of 1846.31 Da (Figure 2A). (Nae-Npe)7 found: 1846.67 m/z [M+1H]1+, 923.69 m/z [M+2H]2+. The 10-mer, GN2-Npm9-C8 was synthesized with 72% purity, confirmed its atomic composition for C91H127N19O10 of 1647.14 Da, and found as: 1647.58 m/z [M+1H]1+, 824.42 m/z [M+2H]2+ (Figure 2B).

Figure 2. LC-MS chromatograms of peptoids, (Nae-Npe)7 (A.) and GN2-Npm9-C8 (B.), showing base peak integration (Left) and the corresponding mass identification (Right).

Conclusion

We have demonstrated the Biotage® Initiator+ Alstra™ microwave peptide synthesizer’s ability to fully automate on-resin synthesis peptoid syntheses. The syntheses included specific reactions for the sub-monomer cycle synthesis of variant peptoids which were achieved with a high degree of crude purity. Furthermore, this was achieved using the interface and tools provided by the software, enabling users to simplify the programming of other peptoid sequences.

Acknowledgements

Associate Professor Biljana Mojsoska, Associate Professor Frederik Dinnes, Post Doctoral Researcher Gustavo Carretero at Roskilde University.

References

1. Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; et al. Peptoids: A Modular Approach to Drug Discovery. Proc. Natl. Acad. Sci. U.S.A. 1992, 89 (20), 9367–9371. https://www.pnas.org/content/89/20/9367  (accessed  2021-07-20).
2. Kirshenbaum, K.; Zuckermann, R. N. Design and Preparation of Organic Nanomaterials Using Self-Assembled Peptoids. Biopolymers 2019, 110 (4), e23265. https://onlinelibrary.wiley.com/doi/full/10.1002/bip.23265 (accessed 2024-03-20).
3.  Robertson, E. J.; Oliver, G. K.; Qian, M.; Proulx, C.; Zuckermann, R. N.; Richmond, G. L. Assembly and Molecular Order of Two-Dimensional Peptoid Nanosheets through the Oil-Water Interface. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (37), 13284–13289.
4. Bicker, K. L.; Cobb, S. L. Recent Advances in the Development of Anti-Infective Peptoids. Chem. Commun. 2020, 56 (76), 11158–11168. https:// pubs.rsc.org/en/content/articlehtml/2020/cc/d0cc04704j (accessed 2024-04-08).
5. Khara, J. S.; Mojsoska, B.; Mukherjee, D.; Langford, P. R.; Robertson, B. D.; Jenssen, H.; et al. Ultra-Short Antimicrobial Peptoids Show Propensity for Membrane Activity Against Multi-Drug Resistant Mycobacterium tuberculosis. Front. Microbiol. 2020, 11, 417.
6.  Mojsoska, B.; Carretero, G.; Larsen, S.; Mateiu, R. V.; Jenssen, H. Peptoids Successfully Inhibit the Growth of Gram Negative E. coli Causing Substantial Membrane Damage. Sci. Rep. 2017, 7, 4235.
7. Diamond, G.; Molchanova, N.; Herlan, C.; Fortkort, J. A.; Lin, J. S.; Figgins, E.; et al. Potent Antiviral Activity Against HSV-1 and SARS-CoV-2 by Antimicrobial Peptoids. Pharmaceuticals 2021, 14 (4), 304. https://www.mdpi.com/1424-8247/14/4/304/htm  (accessed  2021-09-02).
8. Mojsoska, B. Solid-Phase Synthesis of Novel Antimicrobial Peptoids with α- and β-Chiral Side Chains. Methods Enzymol. 2022, 663, 327–340.
9. Mojsoska, B.; Zuckermann, R. N.; Jenssen, H. Structure-Activity Relationship Study of Novel Peptoids That Mimic the Structure of Antimicrobial Peptides. Antimicrob. Agents Chemother. 2015, 59 (7), 4112–4120.
10. Wu, C. W.; Sanborn, T. J.; Huang, K.; Zuckermann, R. N.; Barron, A. E. Peptoid Oligomers with α-Chiral, Aromatic Side Chains: Sequence Requirements for the Formation of Stable Peptoid Helices. J. Am. Chem. Soc. 2001, 123 (28), 6778–6784. https://pubs.acs.org/doi/full/10.1021/ja003154n (accessed 2021-07-20)
     

Supporting information showing the amines used in peptoid sequence assemblies and reagent calculation sheets easily generated by Alstra software upon sequence design.

Amines:

Nae Nce Npe Noc Ntrp Nlys Npm

Sequences:

Peptoid nanosheet positive segment (Nae-Npe)7
PNae PNpe PNae PNpe PNae PNpe PNae PNpe PNae PNpe PNae PNpe PNae PNpe

GN2-Npm9-C8

PNoc PNlys PNtrp PNlys PNlys PNtrp PNtrp PNlys PNtrp PNpm

Reagent calculation

(Nae-Npe)7

GN2-Npm9-C8

Literature number: AN1016