Investigating the Impact of Hydrophobic Polymer Segments on the Self-Assembly Behavior of Supramolecular Cyclic Peptide Systems via Asymmetric-Flow Field Flow Fractionation

The present study examines the behavior of cyclic peptide polymer conjugates that have been designed to combine their self-assembling ability via H-bonding with the properties of amphiphilic diblock copolymers. Using a combination of asymmetric flow-field flow fractionation (AF4) and small-angle neutron scattering (SANS), we have uncovered unique insight based on the population of structures established at a 24 h equilibrium profile. Our results determine that by introducing a small quantity of hydrophobicity into the conjugated polymer corona, the resulting nanotube structures exhibit low unimer dissociation which signifies enhanced stability. Furthermore, as the hydrophobicity of the polymer corona is increased, the elongation of the nanotubes is observed due to an increase in the association of unimers. This encompasses not only the H-bonding of unimers into nanotubes but also the self-assembly of single nanotubes into segmented-nanotube structures with high aspect ratios. However, this influence relies on a subtle balance between the hydrophobicity and hydrophilicity of the polymer corona. This balance is proposed to determine the solvent entropic penalty of hydrating the system, whereby the cost scales with the hydrophobic quantity. Consequently, it has been suggested that at a critical hydrophobic quantity, the solvation penalty becomes high enough such that the self-assembly of the system deviates from ordered hydrogen bonding. The association behavior is instead dominated by the hydrophobic effect which results in the undesirable formation of disordered aggregates.


Polymer-Conjugate Comparisons
Figures S2 shows the comparisons of the amphiphilic conjugates to their control hydrophilic polymers as confirmation of the absence of both one-arm and two-arm unimers.The presence of structures due to the self-assembly of the unconjugated amphiphilic diblock copolymers is also ruled out.This is verified by the differences in the peak elution times and distribution profiles.It is also worth noting that the polymer standards and CP-polymer systems are distinguishable by UV-Vis detection at maxima absorption wavelengths of 220, 280 and 309 nm.The polymers absorb light at 309 nm due to their trithiocarbonate group, while the conjugates absorb at all three wavelengths due to the combination of CP and polymer sensitivity.In all cases, measurements using a UV detector at 280 nm indicated similar distribution profiles as those measured by the RI detector therefore confirming that the populations were conjugate related.

Figures S3 highlights the fractogram comparisons of the amphiphilic conjugates by
their hydrophobic monomers at 20 mol.% hydrophobicity.The results helped assign some of the peaks from the less soluble hexyl acrylate conjugate since quantitative analysis of this conjugate could not be performed.

Method Reproducibility
Figure S5 details example fractograms confirming good method reproducibility hence validating the collected AF4 results and the system performance.Indistinctive differences are observed from the distribution profiles.Precisely, similar peak populations were detected and supplementary calculations (Table S1) further indicated that the structures within the populations are equivalent in size and relative concentrations.Moreover, only slight shifts in the retention times are observed with some conjugates and this can be related to the statistical nature of the AF4 separation process due to inherent fluctuations in the environmental or instrumental conditions.These results are consistent with a recent study by Paul and co-workers who employed molecular dynamics experiments to investigate the influence of different NaCl concentrations on the association of cyclic peptides in water. 1 Their study suggested that at low NaCl concentrations (≤ 0.68-3.42m,~ 0.65-2.85M), the Na + , Cl -ions impact association by screening electrostatic interactions between charged sites on the peptide backbone.Therefore, as our systems' constituent amino acids and polymers do not contain readily ionisable functional groups at the neutral pH of the aqueous NaCl (0.1 M) salt solution, it is logical that the salt does not contribute to the intermolecular association between cyclic peptide units.

∂n/∂c Calculations
Due to the much lower cyclic peptide yield attainable during synthesis (mg), in addition to the higher synthesis costs, the unconjugated polymers were used to estimate the ∂n/∂c of the conjugates based on three general assumptions.The first assumption is that the ∂n/∂c values of the polymers are representative of the conjugates because the polymers make up the majority of the conjugate structure.[8] Lastly, with regards to the amphiphilic diblock copolymers, it was hypothesised that the contributions of each block were proportionately accounted for in the obtained final values. 6,7 his assumption was necessary as the ∂n/∂c of the hydrophobic blocks could not be individually determined in the aqueous solution used for analysis.It is also worth noting that an online approach was implemented as it is fast, straightforward and requires the preparation of less sample in comparison to batch analyses. 9,10 n addition ,the group has validated the accuracy of this approach using common polymer standards whose ∂n/∂c values are available in literature.The mean results obtained from at least 3 consistent measurements are shown in Table S2.

Standard Error (SE) Calculations
The standard error (SE) is a measure of the reliability of the population mean and can be expressed as a percentage, referred to as the relative standard error (RSE).A small SE/RSE (≤ 25%) is an indication that the population mean is an accurate reflection of the samples within a population.

B. SANS
The morphology of the conjugates were evaluated using SANS and Figures S7 (a-c) show reduced scattering data and fits.The insoluble conjugates were not characterised as their propensity to sediment would result in inconclusive data.
With the exception of conjugate (1, CP-[p(DMA)50]2), all featured conjugates were fit to a hairy core-shell cylinder model using SAS view software.The model description is as follows: 12 (,,) = (,)(,) Where CP-[p(DMA)50]2 was fit using a combined model including a core-shell cylinder and Gaussian coil form factor.The remaining conjugates were fit to a core-shell cylinder model.The fits were considered statistically reliable as their Chi 2 values were <20.

Peptide Characterisation Data
Scheme S1: Simplified scheme illustrating the synthesis of the linear octa-peptide (11) using solid phase peptide synthesis (SPPS), the cyclisation of the linear peptide into a cyclic peptide (12) and the Bocdeprotection of the cyclic peptide to allow post-modification (13).

Conjugate Characterisation Data
Scheme S3: Polymer conjugation to the cyclic peptide.R = rest of polymer structure.
FiguresS1 (a-d) below are the conjugate fractograms not included in the main text.The blue and red traces respectively represent signals from the refractive index (RI) and multi-angle light scattering (MALS) detectors.The molecular weight values (right yaxis) for each elution fraction are overlaid as black triangles.

Figure S2 .
Figure S2.Comparison of the studied conjugates to their control polymers via their measured RI detector signals.

Figures S4. 4 (
Figures S4.4 (a-d) are the images of the conjugate solutions where precipitation was observed.

Figure S5 .
Figure S5.CP-[p(BA)5-b-p(DMA)45]2 repeats as an example of good method reproducibility.Repeats of the other conjugates also showed consistency in the resulting fractogram profiles.

2 , 3 5 Figure
Figure S6.AF4-RI traces of example conjugates measured in the aqueous measurement solvent (HPLC grade water) containing 0.1 and 0 M NaCl.

Table S3 :
Relative SE calculations of the molecular weight means from three sample repeats.§ Populations eluting at the specified time, alphabetised populations refer to the nanotube assemblies.*Common units: Da or gmol -1 .† Represents the mean of the values from three sample repeats (population mean).RSE = (SE ÷ x̄p) and SE = (σp ÷√N ); where σp is the standard deviation of the samples within the population, N is the population size and x̄p is the population mean.ConjugateDetermined Using Mean ∂n/∂c (mL/g) SD, σ(mL/g) CP-[p(DMA)50

!Figure S7 :
Figure S7:Comparison of the SANS scattering profiles of the conjugates in 95% D2O and 5% d-DMSO.CP-[p(DMA)50]2 was fit using a combined model including a core-shell cylinder and Gaussian coil form factor.The remaining conjugates were fit to a core-shell cylinder model.The fits were considered statistically reliable as their Chi 2 values were <20.

Figure S9 :
Figure S9: Example SEC trace of a successful chain extension from homopolymer to diblock copolymer.

Figure S14 :
Figure S14: Example SEC trace (DMF) showing the successful conjugation of two polymer arms onto a cyclic peptide and subsequent purification.

Table S1 :
Summary of the size parameters: molecular weight averages (Mw, Mn) and aggregation number (Nagg), of the detected nanotube populations featured in FigureS6.The tabulated values are the means of three data sets/repeats and have low relative standard errors (<5%).
† Population peaking at the specified elution time.*Common units: Da or g mol -1 ; ¤ The detected MALS signals have a low S/N ratio hence calculated weight values omitted due to lower accuracy.‡ Calculated from the weight average molecular weight (Mw); Formula [Nagg = Mw(assembly) ÷ MW(unimer)].

Table S2 :
∂n/∂c values of the conjugates at 25°C in NaCl (0.1M) solution.The RI laser wavelength is within the Infrared-Ultraviolet range.

Table S4 :
Relevant SANS fitting and calculated parameters of the conjugates

Table S7 :
SEC characterisation of the conjugates used in this study.Mw,theo (10 4 ) b,c Mw, SEC (10 4 ) b,c Mn, SEC(10 4) Estimated using ChemDraw software analysis.b Common units = Da or gmol -1 .c Determined by size exclusion chromatography (SEC) calibrated with PMMA standards and using dimethylformamide (DMF) as eluent with 0.1% LiBr additives. a