Beyond Fmoc: a review of aromatic peptide capping groups

Adam D. Martin *a and Pall Thordarson *b

Self-assembling short peptides have attracted widespread interest due to their tuneable, biocompatible

nature and have potential applications in energy materials, tissue engineering, sensing and drug delivery.

The hierarchical self-assembly of these peptides is highly dependent on the selection of not only amino

acid sequence, but also the capping group which is often employed at the N-terminus of the peptide to

drive self-assembly. Although the Fmoc (9H-fluorenylmethyloxycarbonyl) group is commonly used due

to its utility in solid phase peptide synthesis, many other aromatic capping groups have been reported

which yield functional, responsive materials. This review explores recent developments in the utilisation

of functional, aromatic capping groups beyond the Fmoc group for the creation of redox-responsive,

fluorescent and drug delivering hydrogel scaffolds.

Introduction

Self-assembly is a phenomenon which is ubiquitous in nature, from the complementarity of DNA base pairs,1 to the transportof ions across membranes,2,3 the folding of proteins intofunctional tertiary structures,4,5 self-assembly underlies manyof themost fundamental biological processes. The self-assembly ofproteins is of particular interest, as the structure of the protein is intricately linked with its function. Two well-known examples of this are observed in Alzheimer’s disease, where minor modifications to the amyloid and tau proteins result in the self-assemblyand the apparent pathological accumulation of these proteins,resulting in a gain of toxic function.6,7Originally identified as an aggregation-prone region of theamyloid protein, the diphenylalanine sequence has been usedin a number of applications, from semi-conductors to nanophotonics,to optics, with excellent reviews available on thesetopics.8–10 Perhaps one of the most popular applications of the diphenylalanine motif has been its incorporation into shorthydrogel-forming peptides. These hydrogels can be engineeredto mimic the physical and mechanical properties of the extracellular matrix and have been extensively reviewed.11–15 Often, these diphenylalanine containing, self-assembling peptide hydrogels are ‘‘capped’’ at their N-terminus with an aromatic group. The choice of this capping group plays a key role in the subsequent self-assembly of the peptide. It is knownthat the diphenylalanine sequence alone (i.e., NH2-Phe-Phe-OH) will not form hydrogels, instead they tend to form crystalline nanotubes which have excelled in a number of applications.16–18 The introduction of the fluorenylmethyloxycarbonyl (Fmoc) group to the N-terminus of the diphenylalanine sequence (Fmoc-FF), as first reported by Gazit,19 resulted in the formation of a selfsupporting hydrogel, formed through the dilution of Fmoc-FF dissolved in hexafluoroisopropanol with water. Since this initial study, extensive research effort has been applied to elucidating different ways to initiate gelation,20,21 minimizing the variability in resultant hydrogel networks (which are gelation-method dependent),22 and developing applications for these nanostructured  caffolds in tissue engineering, electronics and drug delivery.23–27 .One reason for the popularity of Fmoc-FF in various applications is its ease of synthesis. Fmoc-FF can be synthesised either using solution or solid phase peptide synthesis methods, owing to the common use of the Fmoc group as an amine protecting group in solid phase peptide synthesis (SPPS) and is also commercially available through Bachem. However, this also means that the Fmoc group is susceptible to cleavage at pH values above 10, which can be problematic, as Fmoc-containing peptide gelators are often dissolved in basic aqueous solutions prior to initialising gelation. Upon cleavage of the Fmoc-group from a peptide chain, a highly reactive dibenzofulvalene is formed. While the toxicity of dibenzofulvalene coming from Fmoc-based peptides has not been determined directly, our studies have indicated that Fmoc-FF degradation products show Come cytotoxicity.28 In order to bypass this, a number of different capping groups have been used. The Adams group have popularised the use of a a Dementia Research Centre, Department of Biomedical Science, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia.

E-mail: adam.martin@mq.edu.au

b School of Chemistry, The Australian Centre for Nanomedicine and the ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, University of New South Wales, Sydney, NSW 2052, Australia.

E-mail: p.thordarson@unsw.edu.au

Received 10th November 2019,

Accepted 8th January 2020

DOI: 10.1039/c9tb02539a

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naphthalene-based capping group,29–32 which has the advantages

of not being base-labile and boasts several sites for additional

functionalisation. Other popular capping groups which have

previously been reviewed include the carboxybenzyl and cinnamoyl

groups,33–35 which due to their decreased aromaticity,

require hydrophobic peptide sequences such as diphenylalanine

to form hydrogels. The photoresponsive spiropyran, azobenzene

and dansyl-based capping groups have also previously been

reviewed.36

The alteration of the N-terminal capping group sits alongside

other strategies such as selection of peptide sequence and

gelation method in facilitating the tuning of peptide self-assembly

and the properties of the resultant hierarchical structures. This

review is not exhaustive but will selectively focus on recent progress

made in expanding the chemical diversity of moieties which

have been used to cap the N-terminus of short aromatic

peptides. In addition to broadening the chemical landscape

available to researchers, these new capping groups have yielded

insights into the design rules which govern the self-assembly of

short peptides into hydrogels, whilst concomitantly generating

new functional materials.

Heterocyclic capping groups

Heterocycles are abundant in nature (i.e., many drug molecules,

the amino acid tryptophan, nucleic acids) and are a good

starting point for expanding the chemical diversity of the

N-terminal capping group in short peptides. As early as 2012,

the capping of a pentapeptide Ala-Gly-Ala-Gly-Ala (AGAGA)

sequence with an ex-tetrathiofulvalene (exTTF) was reported.37

In this example, the TTF group is positioned either side of the

9- and 10-positions of an anthracene group, resulting in a

deviation from planarity. This, associated with the hydrophobicity

imparted by the anthracene group, yielded the formation

of helical nanofibers where the TTF units do not

interact with each other. These nanofibers formed only in

halogenated organic solvents, with ageing behaviour (large

bathochromic shifts in absorption spectra) observed in methylcyclohexane

that could be reversed through the addition of

methanol.

In 2014 Ulijn et al. attached a TTF group to a diphenylalanine

peptide bearing an amine at its C-terminus (TTF-FF-NH2, Fig. 1a)

and observed gelation in several organic solvents including

chloroform, ethyl acetate, DMSO and tetrahydrofuran.38 Once

gelation was established, the peptide was mixed with the acceptor

tetracyano-p-quinodimethane (TCNQ) and iodine vapour to yield a

supramolecular charge-transfer organogel, with characteristic

peaks observed for TCNQ__ and TTF_+ species. Drop casting

of the organogel between gold contacts revealed a significant

increase in conductivity upon incorporation of TCNQ, suggesting

the successful generation of charge-transfer supramolecular nanofibers

(Fig. 1b).

By switching from TTF group to naphthalene diimide (NDI),

Ulijn et al. was able to form these charge-transfer supramolecular

nanofibres in aqueous environments.39 This was

achieved through a biocatalytic pathway whereby the enzyme

thermolysin was used to condense naphthalene diimide-tyrosine

(NDI-Y) with phenylalanine amide (F-NH2) in the presence of

dialkoxynaphthalene derivatives, either 1,5-dialkoxynaphthalene

(1,5-DAN) or 2,6-dialkoxynaphthalene (2,6-DAN). Upon the addition

of the electron rich DAN donors, to the electron poor NDI-Y, a

highly coloured charge-transfer complex was observed, corresponding

to spherical aggregates. The addition of thermolysin

induced the formation of a highly coloured hydrogel composed

of charge-transfer nanofibres, which were imaged using AFM and

charge-transfer confirmed using fluorescence spectroscopy.

Naphthalene diimides have also been used by Lin et al. to

create multifunctional compounds which can be used for cell

imaging or self-assemble into hydrogels at higher concentrations