These indirect measurements serve to illustrate that small changes in aliphatic branching can modify the intramolecular dispersive interactions of each species. We also investigated leucine and valine bases (Leu-, Val-NH2), and well-defined IFC sheets and microscopic terraces were observed for both systems (Figure and). Of the two, Leu-NH2 formed IFC structures most readily, with behavior like Nab and Nle, confirming that a higher dispersive attraction does promote IFC but also that the sidechain dispersion of Nl was not a limiting factor. An additional confirmation of this being that the calculated LogP for the valine is in fact lower than Nl (Table ). Taken together these data allow us to establish that dispersive attraction does not clearly correlate with IFC propensity. ATR-FTIR showed distinct absorbance's in both the carbonyl stretch and N-H bending regions for both Leu-NH2 and Val-NH2 when comparing to peptoid bases (Figure ). This indicated that the specific nature of the hydrogen bonding network does not directly impact the IFC phenomena. This is in contrast to past studies comparing the self-assembly of Fmoc-F and its peptoid analogs, for which different morphologies were attributed to differences in hydrogen bonding capacity. Sidechain Architecture, Conformation and the Importance of 𝝌 𝟐 Torsion It is well-known that small modifications in sidechain design can have a profound impact on resultant nanoassemblies. The dominant cause of intrinsic β-sheet propensity of amino acid residues in proteins has previously been attributed to the avoidance of steric sidechain and backbone clashes. We hypothesized that a similar mechanism is operative in IFC packing, where sidechains and backbones are optimally segregated into hydrophobic and ionic layers. In the case of the amino amides, the sidechain is perpendicular to the backbone, which we expect to assist packing optimization. While in peptoids, the sidechain is parallel to the backbone, furthermore the IFC peptoids Nab and Nle both share a linear fragment of two consecutive -CH2-units, which is lacking in Nl. We speculated that the longer ethylene linkage may provide a degree of flexibility to optimize hydrogen bonding, dispersive and ionic interactions within a crystal lattice. To assess the impact of this on conformation we performed 𝜒 torsion scans at the MP2/6-31G(d) level of theory for each peptoid base. A common feature between all species were conformational minima at ± 60 o and ± 180 o (Figure ). However, for Nl significant energy barriers existed between the 60 o and -60 o , 180 o torsions (~ 5.5 kcal/mol) and the global minima was -6.88 kcal/mol. In contrast for both Nab and Nle, these barriers were smaller, the energy profiles more symmetric and the global minima for Nab (the isomer of Nl) lower at -8.66 kcal/mol. Sampling of the 𝜒 in 100 ns MD simulations of Nl, Nle and Nab in ACN with chloride anions agreed with this finding: with significant populations of ± 180 o and -60 o for Nl and much less sampling of 60 o (Figure ); whereas Nab/Nle sample both ± 60 o and ± 180 o in a broadly symmetric manner (Figure ). The explanation for this lies in the proximity of the isopropyl unit and backbone. The isopropyl Cβ unit has two -CH3 groups and a proton; the observed significant energy penalties in the QM torsion scan arise when the methylene units and the backbone a close, whereas this is significantly reduced for the proton (conformers with red and black steric clashes respectively, Figure ). The consequence being that the energy landscape of 𝜒 is asymmetric, with the energy associated with the transition -60 o ↔ -180 o being is much less than 60 o ↔ 180 o . Consequently, two different populations of conformation will exist within a sample of Nl, which broadly speaking is not the case for Nab/Nle. In these species the two consecutive -CH2-units will interact with the sidechain in a lower energy and symmetric manner. This enables sufficient spatial separation between sidechain bulk and the backbone, lowering the transition energies -60 o ↔ -180 o /60 o ↔ 180 o and resulting in a purer conformational ensemble. Elsewhere, Nl and Nle residues were incorporated into peptoid nanosheet forming 28mer sequences as (Nke-Nx)7 -(Ne-Nx)7, where Nx = Nl or Nle. Here, the nanosheets formed by Nl were found to be insufficiently stable for characterization and this was attributed to low hydrophobicity. In light of our findings, a contributing factor to this may also have been the conformational inflexibility of Nl, which could disrupt packing of the hydrophobic core and destabilize a nanosheet assembly. Noteworthy, the oligomer sequence containing Nle also showed a tendency to self-stack which is another interesting parallel with our current study. To investigate further the internal structure of Nab.HBr and Nle.HBr we obtained single crystal X-ray diffraction structures for these species. This revealed both to be composed of successive hydrophobic and ionic layers interdigitated on the monomer scale (Figure and). The former being composed of aliphatic sidechains from neighboring layers facing each other. The latter consisting of 2:1 coordination between bromide anions and an N-terminal amine and an amidated C-terminus from adjacent monomers, which result in a continuous linkage of stacked monomers. The structure is further reinforced by hydrogen bonding between consecutive layered C-terminal amide groups, which corroborates the FTIR data (Figure ). The overall stacked/layered structure is also in agreement with the terraced morphology observed in optical microscopy, AFM and SEM images. While there is a similarity to the organization of neutral aliphatic amino acids within their crystals formed in the bulk, but these structures instead have 'hydrophilic' layers. Notably the inclusion of the amidated amine group in bromide coordination confirms that this C-terminal functionality is important for IFC and is present in all salts within our study. Furthermore, 𝜒 is ~ 180 o for both species, which corroborates both energy minima at the MP2/6-31G(d) level of theory and sampling in MD simulations, where this torsion constitutes the highest occupancy for both species. Noteworthy, given the amorphous/semicrystalline macroscopic nature of Nl assemblies we did not attempt to obtain a single crystal X-ray diffraction structure for this salt. Taken together, we propose that IFC formation does not occur for Nl as the growth of well-defined structures would be impeded by conformational heterogeneity (Figure ). This goes to explain by Nl crystals have a different morphology, transparency and appear softer, as well as why this species has a distinct hydrogen bonding network. Furthermore, it follows that the similarity in 𝜒 profiles for Nab and Nle account for their observed similarity in IFC behavior. As such, an apparent molecular design principle for aliphatic peptoid IFC molecules is that they possess sufficient steric freedom around the 𝜒 torsion. Anion Selectivity, Coordination and Migration in IFC Growth Process With the X-ray crystal structure of Nle.HBr as an endpoint and taking into consideration the packing organization promoted by the ionic core, we hypothesized that ion migration to the interface via poor solvent affinity must drive IFC growth. From here, templated growth occurs by coordination of cationic amines and the interdigitation of aliphatic sidechains. To test this proposed mechanism, we decided to screen different anion types with variable solvent affinity to see how this affected crystallization. In examining the role of the anion, and thus further probing the generality of the IFC phenomenon, we focused on Nle. The anions valerate, acetate, sulfate, and chloride were selected. Apart from the chloride, all other anions were expected to have greater affinity for ACN solvent as they can dissociate charge across multiple atoms which would improve compatibility with apolar solvent. Indeed, as shown in Figure , no growth was seen for either valerate or acetate salts, nor for the sulfate anion. In contrast, the halide salts provide anions with comparatively more charge localization, and they all gave rise to IFC (Figure ). Similar crystal morphologies were observed for both Nle.HCl and Nle.HBr (Figure -41). To further confirm the alternating layer structure, which would concentrate anions at successive interfacial planes, we performed time of flight secondary ionization mass spectrometry (TOF-SIMs) under conditions that sputtered ions only within a depth of 1~2 nm. Figure 4B indeed shows that the surface of an Nle.HBr crystal contained a substantially higher abundance and homogenously distribution of bromine and hydrogen isotopes, verses carbon, oxygen, and nitrogen. This showed that atoms belonging to the ionic layer and those belonging to the hydrophobic layer (i.e., the bulk of the peptoids) were indeed segregated. To build on this notion of differences in anion solvent affinity, and presumed differences in anion denticities, e.g., acetate charge is delocalized, unlike in the halide anions and the importance of this to IFC we performed MD simulations to compare the aggregation of Nle in the presence of either ion. Visually, we observed that Nle.HCl forms a more packed structure than Nle.H-acetate (Figure ). These observations are consistent with an increased ACN solvent affinity for the acetate anion. Radial distribution functions (RDFs) were also obtained to compare coordination between a given anion as the N-terminus (NP) or amidated C-terminus (NT) as informed by the crystal structures we obtained. To ensure an equal number of sites were used, we calculated the acetate RDF using the carboxylate carbon in the acetate anion. For the Nle.HCl we obtained 2:1 coordination between NP and NT centers, which is in good agreement with the obtained X-ray crystal structures for Nle.HBr. In contrast, for the acetate, the ratio was 1:1 in NP and NT (Figure ), with a much lower number of contacts as consistent with the visibly amorphous ionic core. These findings confirm that anion coordination, and associated solvent affinity play a crucial role in the viability of the IFC phenomenon. Based on our observations, we note a striking resemblance between IFC packing structures and those for much longer anionic-aromatic peptoid sequences reported by Ma et al. In their solid-liquid system, which represents another example of 2D interfacial growth, where crystalline assembly is templated on mica by amphiphilicity arising from the sequence design, supported by sidechain carboxylate-calcium coordination and aromatic non-covalent dispersive interactions. Thus, the present monomer system highlights that pre-organization of non-covalent interactions along peptidic sequences and aromatic functionality are not required for interfacial assembly.