Self-Assembly, Nematic Phase Formation, and Organocatalytic Behavior of a Proline-Functionalized Lipopeptide

Abstract

The self-assembly of the amphiphilic lipopeptide PAEPKI-C16 (P = proline, A = alanine, E = glutamic acid, K = lysine, I = isoleucine, and C16 = hexadecyl) was investigated using a combination of microscopy, spectroscopy, and scattering methods and compared to that of C16-IKPEAP with the same (reversed) peptide sequence and the alkyl chain positioned at the N-terminus and lacking a free N-terminal proline residue. The catalytic activity of these peptides was then compared using a model aldol reaction system. For PAEPKI-C16, the cryo-TEM images showed the formation of micrometer-length fibers, which by small-angle X-ray scattering (SAXS) were found to have radii of 2.5–2.6 nm. Spectroscopic analysis shows that these fibers are built from β-sheets. This behavior is in complete contrast to that of C16-IKPEAP, which forms spherical micelles with peptides in a disordered conformation [ Hutchinson Hutchinson J. Phys. Chem. B2019123613]. In PAEPKI-C16, spontaneous alignment of fibers was observed upon increasing pH, which was accompanied by observed birefringence and anisotropy of SAXS patterns. This shows the ability to form a nematic phase, and unprecedented nematic hydrogel formation was also observed for these lipopeptides at sufficiently high concentrations. SAXS shows retention of an ultrafine (1.7 nm core radius) fibrillar network within the hydrogel. PAEPKI-C16 with free N-terminal proline shows enhanced anti:syn diastereoselectivity and better conversion compared to C16-IKPEAP. The cytotoxicity of PAEPKI-C16 was also lower than that of C16-IKPEAP for both fibroblast and cancer cell lines. These results highlight the sensitivity of lipopeptide properties to the presence of a free proline residue. The spontaneous nematic phase formation by PAEPKI-C16 points to the high anisotropy of its ultrafine fibrillar structure, and the formation of such a phase at low concentrations in aqueous solution may be valuable for future applications.

Multivalent display of chemical signals on self‐assembled peptide scaffolds

“…[ 347 ] This sequence was inverted to yield the peptide amphiphile PAEPKI‐C 16 (Figure 13), which possesses a free N‐terminal proline residue. [ 175 ] This peptide amphiphile was shown to form β‐sheet fibrous assemblies in water that can further entangle to form hydrogels above 5 wt%, which contrasts the formation of spherical micelles observed for C 16 ‐IKPEAP. While both assemblies could catalyze the aldol condensation of p ‐nitrobenzaldehyde and cyclohexanone, PAEPKI‐C 16 displayed enhanced diastereoselectivity under all conditions tested and higher conversion in most cases, implicating accessibility of the free N‐terminal proline as a critical component to afford selectivity in asymmetric aldol catalysis.…”

“…B, Representative examples of selfassembling peptide materials that act as aldolases inverted to yield the peptide amphiphile PAEPKI-C 16 (Figure 13), which possesses a free N-terminal proline residue. [175] This peptide amphiphile was shown to form β-sheet fibrous assemblies in water that can further entangle to form hydrogels above 5 wt%, which contrasts the formation of spherical micelles observed for C 16 Das and coworkers developed self-assembled peptide amphiphile nanotubes for forward and retro-aldol catalysis that leveraged both lysine and tyrosine for catalytic activity. [176] The peptide amphiphile C 10 -FFVK ( Figure 13) initially self-assembled to form nanofibrous assemblies, but these fibers further associated as the sample aged to form nanotubes with solvent-exposed lysine residues on the surface.…”

“…Ac-K 4 (ChaKChaE) 2 -NH 2 Lysine residues 3C [153] Ac-K 2 (ChaKChaE) 2 -NH 2 Lysine residues 3C [153] Ac-E 4 (ChaKChaE) 2 -NH 2 Glutamic acid residues 3C [153] Ac-E(CH 2 CHO)EEEAAAVVVK(C 16 )-NH 2 Silver nanoparticles 3C [154] Fmoc-FFECG Silver nanoparticles 3C [155] C 16 -V 3 A 3 K 4 -NH 2 Folate 3D [156] CKKAAVVK-C 12 KLVWLPK-NH2 3D [157] FFERGD 10-hydroxycamptothecin (HCPT)/cisplatin 3D [158] (VPGVG) 80 (VPGSG) 60 EgA1 3D [159] Nap-G D F D F Phosphate/YSV 3D [160] PhAc-FFAG LDD 3D [16] LVFFGFLG RGD/camptothecin 3D [161] Ac-IHIHIQI-NH 2 Histidine residues 3E [162][163][164] Ac-IHIHIYI-NH 2 Histidine residues 3E [163] Im-KLVFFAL-NH 2 Lysine residues/imidazole groups 3E [165] Ac-KLVFFAL-NH 2 Lysine residues 3E [166] Sialic acid 3F [167,168] AQZ/near-infrared quantum dots/Alexa Fluor 633 3F [169] Ac-HLVFFAL-NH 2 Histidine residues/hemin 3E [170] VK2H (HSG(VK) 4 V D PPT(KV) 4 -NH 2 ) Histidine residues 3E [171] Phenylalanine Zn 2+ 3E [172] 1,4,5,8-naphthalenetetracarboxylic acid diimide Proline residues 3E [173] PFE-C 12 Proline residues 3E [174] PEF-C 12 Proline residues 3E [174] PAEPKI-C 16 Proline residues 3E [175] C 10 -FFVK Lysine residues/Fmoc-Tyrosine 3E [176] Ac-LHLHLQL-NH 2 Histidine residues/hemin 3E [177] Ac-LHLHLFL-NH 2 Histidine residues/hemin 3E [177] Ac-LILHLFL-NH 2 Histidine residues/hemin 3E [177] HN-D PL-CONH-C 16 D-proline/leucine residues/IrCp* 3E [178] KKAAVV(K)-C 12 RTL (RTLAFVRFK) 3F [179] SFEEA...…”

“…[173] PK-NDI self-assembled in a β-sheet-like Several self-assembling peptide amphiphiles have also been developed that display aldolase activity. [174][175][176] Two tripeptides, PFE and PEF, were conjugated to hydrocarbon chains to yield peptide amphiphiles PFE-C 12 ( Figure 13) and PEF-C 12 , respectively, that selfassembled to form nanofibrillar aggregrates with the N-terminal proline displayed on the surface. [174] Quantitative conversion of p-nitrobenzaldehyde and cyclohexanone to the aldol condensation product was afforded by aggregates of both PFE-C 12 and PEF-C 12 after 72 h, though quenching after shorter reaction times revealed that PFE-C 12 possessed higher aldolase activity than PEF-C 12 .…”

“…Hamley and coworkers also recently developed a self‐assembling peptide amphiphile system displaying proline to affect aldol catalysis. [ 175 ] C 16 ‐IKPEAP, a lipid conjugate of the N‐terminal hexapeptide fragment from the human gut hormone PYY 3‐36 , has recently been shown to assemble into spherical micelles in aqueous media, but form β‐sheet fibrillar structures when dried. [ 347 ] This sequence was inverted to yield the peptide amphiphile PAEPKI‐C 16 (Figure 13), which possesses a free N‐terminal proline residue.…”