Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Summary

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Abstract

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Introduction

Self-assembling peptide amphiphiles (PAs) are a class of biomaterials typically comprised of the following segments: (a) hydrophilic head, (b) linker region, and (c) hydrophobic tail. Most PAs described in the literature possess a hydrophilic head comprised of charged or polar amino acid residues^1,2,3,4. PAs have found a wide range of applications in biomedicine including drug delivery, disease diagnostics, regenerative medicine, etc.⁵. Based on their amino acid sequence, PAs can form a wide variety of nanost....

Protocol

1. General Protocol for Synthesis of PPAs

1. Calculate the scale of synthesis (usually mmol). This scale is based on the mass of the target PPA amount. Keep in mind that the reaction efficiency of SPPS gradually decreases with an increase of amino acid sequence. Therefore, the exact reaction efficiency is difficult to calculate.
2. Calculate the weight of the resin to be used according to the loading of the resin. The loading is found on the container or the analysis protocol of the resin and expressed in mmol/g. The following formula can be used for calculating the weight of the resin:
3. Carefully weigh out 2-chlorotrityl chloride resin (in ou....

Results

After the synthesis and purification and before physicochemical or biological evaluation, it is recommended the masses of the PPAs are re-checked and the purity ascertained using analytical HPLC. For material characterization or biological evaluation, PPAs need to have a purity of >95%. Figure 2 shows the HPLC trace (Top) and MALDI spectra (Bottom) confirming the presence of the product. HPLC analytical systems will integrate the area under the curve (AUC).......

Discussion

The protocols described here can be used to synthesize PPAs as wells as PAs and related peptide-based molecules (such as hybrid PA-peptoids). Although the synthesis of peptides using SPPS is a straightforward procedure, the synthesis of peptides containing biological homing molecules can be particularly challenging. Polyamines like spermine, spermidine, diethyelenetriamine, etc., can function as homing molecules for targeting cancer cells¹³. The PPAs can self-assemble into nanostructures .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Chlorotrityl chloride resin	AappTec	RTZ001
SynthwareTM synthesis vessel	Aldrich	SYNP120050M
Dichloromethane	Acros	AC406920250	Fisher Sci. Catalogue #
Wrist Shaker	Boekel Scientific	401000-2
Kaiser test kit	Sigma-Aldrich	60017
2-[(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-amino]-ethanol	Sigma-Aldrich	CDS004772
Anhydrous Methanol	Acros	AC610981000	Fisher Sci. Catalogue #
Chloranil test kit	TCI	TCC1771-KIT	VWR Catalogue #
Di-tert butyl di-carbonate	Acros	AC194670250	Fisher Sci. Catalogue #
Dimethylformamide	Fisher Scientific	BP1160-4
Hydrazine	Acros	AC296815000	FIsher Sci. Catalogue #
(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)	p3biosystems	31001
4-methyl piperidine	Acros	AC127515000	FIsher Sci. Catalogue #
Trifluoroacetic Acid	AappTec	CXZ035
Triisopropyl Silane	Sigma-Aldrich	233781
Ether	Fisher Scientific	E138-1
α-Cyano-4-hydroxycinnamic acid	Sigma-Aldrich	C8982
9-Aminoacridine	Sigma-Aldrich	92817
Fisherbrand Syringe Filters: PTFE Membrane	Fisher Scientific	09-730-21