Do peptide barrels allow SWCNT chirality enrichment?

The manuscript in section 4.3.1 shows, that it is possible to useaHBs for the dispersion of SWCNTs. Their predefined pore diameter should - in principle - also allow for enrichment of certain SWCNT chiralities/diameters. The AFM experiment shown in Fig. 3 (p. 88) also suggests, that there is a difference even when comparing the two aHBs with their different pore diameters. Whereas the B2-barrel (d = 7.6 Å) shows alternating heights corresponding to peptide barrels wrapping (6,5)-SWCNTs (d= 7.6 Å), the control sample with the barrel C2 shows ’naked’ tubes with a mean diameter of 5 Å. These could, amongst others, correspond e.g. to the smaller metallic (5,2)-SWCNTs with a diameter of 5 Å, which would not be detectable in the absorbance spectra due to their metallic nature. In this case, however, the C2 peptide would have been desorbed under the

Figure 4.1.: Fitted NIR absorbance spectra of different SWCNT samples. a) NIR-absorbance spectrum of (GT)20-DNA dispersed SWCNTs and the corresponding fit. b) absorbance spectrum of a SWCNT/SDBS sample and the corresponding fit. c) NIR-absorbance spectrum of SWCNT@B2 and the corresponding fit. d) Comparison of the relative peak areas of the three different samples for four SWCNT-chiralities.

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same washing procedure applied also to the SWCNT@B2 sample indicating a weaker C2-SWCNT-binding. Fig. 4.1 shows the background subtracted (e ^b ) NIR absorbance spectra of carbon nanotubes dispersed using (GT)20-DNA, the B2aHB and the surfactant SDBS fitted with nine Lorentzians corresponding to different SWCNT-chiralities. The barrel B2 does not exclusively disperse a single SWCNT-chirality, but does show a trend towards encapsulating smaller SWCNT diameters compared to the SWCNT/(GT)20 and SWCNT/SDBS sample (e.g. for (6,5)-SWCNTs: 48% ((GT)20), 62% (B2), 37% (SDBS)).

From the combination of AFM and NIR absorbance spectroscopy one can conclude, that the peptide barrels can ’breathe’ and adjust to encapsulate different chiralities, yet still preferentially binds those with exactly fitting diameters.

While these AFM studies are not representative for the whole sample and NIR absorbance spectroscopy does only show the semiconducting SWCNTs, as a next step also Raman-spectroscopy should be employed to get a better understanding of all involved chiralities characterized by their radial breathing modes (RBM). In future studies, one could also combine the preferential binding with enrichment techniques such as aqueous-two-phase (ATP) separation^[190] to useaHBs for single-chirality isolation.

4.3.3. Discussion

Up to now, peptides were - despite their great versatility - only rarely utilized for the mod-ification of carbon nanotubes. For comparison, a PubMed search with the terms "carbon nanotube AND peptide" in the title/abstract yielded 121 results compared to 492 results for "carbon nanotube AND DNA". In general, the reported studies can be categorized into three distinct approaches.

First, the use of phage-displayed peptide libraries to find peptide sequences with a high affinity for carbon nanotubes, which led to the peptides containing conserved hydropho-bic/aromatic residues found by Wang et al.^[191] and Su et al.^[192]. In a follow-up study, Su et al. found especially the amino acid tryptophan crucial for effective nanotube binding.^[193]Second, peptides were also conjugated covalently to SWCNTs leading to de-stroyed NIR fluorescence. This approach includes mainly examples, where the SWCNTs were used e.g. as delivery vehicles for bioactive peptides to cross cellular membranes^[80]

or as shielding agents preventing the generation of immune responses against the conju-gated peptide.^[194] Third, researchers made use of special peptide characteristics such as amphiphilicity, reversible cyclization or the formation ofa-helices on SWCNTs for the

dis-4.3. Peptide barrels as a novel functionalization platform for SWCNTs out by Dieckmann et al. in 2003, who used a peptide sequence they termed nano-1. This peptide was then shown to ab able to fold into ana-helix in the presence of SWCNTs lead-ing to their dispersion.^[120,195] In 2005, Arnold et al. used an anionic peptide amphiphile (containing an alkyl-tail for SWCNT-binding) termed PA3 for SWCNT dispersion^[196], while Ortiz-Acevedo et al. showed the possibility of using reversible cyclic peptides for reversible SWCNT dispersion (upon disulfide formation/cleavage).^[121] In 2011, Grygo-rian et al. further expanded on the idea of Dieckmann et al. with a computational design approach addressing a variety of different parameters such as residues interacting with the SWCNT, the pitch angles of different SWCNT chiralities and the feasibility of pep-tide design and stability. Using these parameters, they found two sequences, which form a-helical coiled-coil hexamers with parameters matching the geometry of (3,8)-SWCNTs and were also able to show that they can enrich, yet not exclusively bind this chirality.

Furthermore, they also used cysteine-residues introduced close to the N-terminus for the templated attachment of gold nanoparticles.^[122]

In contrast to these examples of peptides encapsulating carbon nanotubes, this work makes use of previously characterized, de novo designed a-helical coiled-coil barrels.

While the work of Grygorian et al. is a truly impressive example of what computational peptide design can yield, it is a clear advantage to be able to rationally ’choose from a catalogue’ of aHBs with different diameters^[114] rendering the whole procedure a very straightforward process. The fact, that aHBs self-assemble in aqueous solution and are very well characterized themselves in terms of sequence-structure relationships is crucial for this attempt to gain also further use in other applications. Figure 2 (p. 88) shows the difference in dispersion yield for the four peptides chosen - two with a larger (B1/B2, d= 7.6 Å) and two control peptides with a very narrow pore (C1/C2,d= 3.9 Å). While the two peptides containing the larger pore were able to disperse SWCNTs, the C1/C2 peptide were not successful. But also the C-terminal truncation of B1 leading to B2 led to a significant increase in dispersion yield, which could be attributed to a reduced sta-bility of the aHBs on their own leading to increased assembly on the nanotube and thus enhanced dispersion. While it is important to note that the pore diameters estimated from the X-Ray structures of the barrels themselves are not fixed values, but can rather

’breathe’, Fig. 3 (p. 88) as well as section 4.3.2 show a trend towards smaller SWC-NTs being enriched with the smaller pore peptides (C1/C2) compared to B1/B2. This is especially apparent in the AFM images shown in Fig. 3. Whereas this could also be attributed to larger adsorptive forces of the C2-peptide on mica, it is more likely that

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the smaller measured heights (approx. 0.5 nm) are due to a more stable dispersion of smaller SWCNT species. The smallest free-standing SWCNT reported to date has a di-ameter of 0.43 nm.^[7] However, in the absorbance as well as fluorescence spectra with excess peptide present in the solution, this difference is not as apparent as in the AFM images. This could also be due to the tubes in the AFM image being mainly smaller di-ameter, metallic tubes which are not visible in the optical signature of the sample. These assumptions will be subject of further study including techniques such as Raman spec-troscopy.

The next important step is now to further increase the colloidal stability of the SWCNT@

Barrel hybrids to the point, where no excess peptide in solution is necessary anymore.

This in turn would enable functionalization of outwards-facing residues (e.g. f-positions, compare Fig. 2.12) with fluorophores or targeting-units. It is, however, important to note, that this was also not accomplished in all other approaches mentioned above em-phasizing the need for novel techniques for stabilization. In preliminary experiments Jan Horlebein (in his B.Sc. thesis) achieved together with the author of this thesis a slight stabilization of the hybrid structures towards removal of excess peptide by utiliz-ing so-called peptide nanotubes (PNTs) instead of sutiliz-ingle aHBs. These structures based on aHBs with ’sticky ends’ were recently described by the Woolfson lab^[118,119] to form tubular structures of several micrometers in length and could thus also possibly lead to a more densely covered SWCNT surface and thus increased colloidal stability. In addi-tion, also carbodiimide-initiated cross-linking or native chemical ligation could be used to covalently link adjacent barrels to one another fixing the structures and preventing disassembly upon removal of excess peptide. While these approaches will be subject of further study to generate a stable peptidic platform for carbon nanotube functionaliza-tion, the work described in the following section will employ sp³defects for the covalent attachment of peptides and proteins with retained optoelectronic properties.