| HPLC reaction monitoring (1.0 mM azido-peptide, 1 eq. CuSO₄, 4 eq. ascorbic acid, 4 eq. alkyne) | Each combination showed an immediate decrease in the azido-peptide HPLC peak within the first five minutes, accompanied by a new peak at a higher retention time, indicating that the newly formed species is more hydrophobic than its reactant; time-dependent analysis revealed monoexponential consumption of the starting material in all cases; equilibrium conversions of 35–60 %; all combinations reached equilibrium within approximately 20 minutes |
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| DiO-C₆ fluorescence assembly monitoring | C₆- and C₈-derived peptide amphiphiles rapidly assembled in contrast to their C₁₂-derived counterparts; the length of the alkyl spacer in the peptide precursor exerted negligible influence on the assembly rates; t₁/₂ ≈ 35 minutes for the shorter hexyne and octyne segments |
| Circular dichroism spectroscopy | Almost all derivatives exhibited a characteristic random-coil signature; P₈C₁₂ showed β-sheet character |
| Confocal fluorescence microscopy after 5 h — C₆- and C₈-based peptide amphiphiles | Isotropic colloidal morphology of micrometer-sized particles |
| Confocal fluorescence microscopy after 5 h — P₆C₁₂ and P₈C₁₂ | Well-defined fibers with a strong tendency to form hierarchical structures via bundling |
| Confocal fluorescence microscopy — P₂C₁₂ | Irregular architectures after 5 h; transitioned into fiber bundles matching those of P₆C₁₂ and P₈C₁₂ after 24 h |
| Confocal fluorescence microscopy after prolonged aging — P₂C₆ and P₂C₈ | Fibrous morphology remained inaccessible |
Nils Bäumer, Monika Wenisch, Ignacio Insua, Job Boekhoven. “Peptide amphiphile-based organelles for fueled synthetic cells.” ChemRxiv (). https://doi.org/10.26434/chemrxiv.15007627/v1
The main paper refers to supporting information, but a separate supporting information file was not found among the files available from the ChemRxiv record
| HPLC phase partitioning (EDC 75.0 mM; Ac-FRGRGD-OH 12.3 mM; pSS 4.3 kDa, 40.0 mM; 1 min after EDC addition; centrifugation for 5 min at 10,000 rpm) | HPLC was used to quantify peptide amphiphile concentrations in the droplet and supernatant, allowing calculation of the partitioning coefficient; all partitioning coefficients indicated highly preferential accumulation of peptide amphiphile assemblies in the droplets, with a gradual increase as tail length increased |
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| Fluorescence microscopy (EDC 75.0 mM; Ac-FRGRGD-OH 12.3 mM; pSS 4.3 kDa, 40.0 mM; control: fuel-dependent synthetic cells without peptide amphiphile assemblies) | Early in the lifecycle, the droplets appeared almost identical to the control; the droplets continued to fuse, forming larger droplets; vacuolization resulted in spherical shells; irregular protrusions and thicker sections occurred in the shell structures; the observation was independent of peptide amphiphile molecular composition |
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| Fluorescence microscopy (EDC 75.0 mM; Ac-FRGRGD-OH 12.3 mM; pSS 4.3 kDa, 40.0 mM) | Independent droplets formed and did not interact with the hierarchical peptide amphiphile fibers |
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| FRAP (EDC 75.0 mM; Ac-FRGRGD-OH 12.3 mM; pSS 4.3 kDa, 40.0 mM; NBD-labeled RG₂N 0.5 µM; control: fuel-dependent synthetic cells without peptide amphiphile assemblies; 3 technical replicates of 3 chemical replicates, n = 9) | Lower diffusivity in the kinetically trapped state compared to the control |
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| Fluorescence microscopy (7, 15, and > 20 min; NBD-G(RG)₂N-NH₂ and Cy5-labeled pSS derivative, 404 kDa) | Round, homogeneous droplets fused into larger structures over time; towards the end of the lifecycle, vacuolization produced uneven spherical shells |
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| Fluorescence microscopy (7, 15, and > 20 min; NBD-G(RG)₂N-NH₂ and Cy5-labeled pSS derivative, 404 kDa) | Within a few minutes, bright speckles appeared in the NBD-G(RG)₂N-NH₂ channel, indicating preferential accumulation of the peptide in a discrete domain within the coacervate; the same regions appeared as dark holes in the pSS channel, indicating exclusion of pSS; the domains matured into discrete assemblies resembling those formed by P₂C₁₂ after several hours of equilibration in isolation, indicating a secondary phase-separation of the in situ formed peptide amphiphile; pSS exclusion occurred before droplet vacuolization began; these discrete assemblies were never observed being expelled from the compartment altogether, nor fully exposed to either the vacuoles or the surrounding dilute phase |
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| Fluorescence microscopy (phase separation with Ac-FRGRGN-NH₂) | Structural transition from discrete to fibrous assemblies that can grow to lengths of up to several microns within about 1 h; fibrillar structures in isolation took upwards of 5 h to form; this structural transition preferentially occurs in larger droplets |
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