Sequence Engineering Overrides Side-Chain Stereochemistry in Governing the Thermal Stability of Polyproline Copolymers

Abstract

Recent advances in N-carboxyanhydride (NCA) synthesis and controlled ring-opening polymerization have enabled the precise preparation of sequence-engineered polypeptides with programmable structures and functions. This study examines the influence of sequence distribution and side-chain stereochemistry on the thermal stability and aggregation behavior of polyproline-based copolymers. A series of statistical and diblock copolymers was synthesized by ring-opening copolymerization of L-proline N-carboxyanhydride (LP-NCA) with either L-proline-(4R)-propargyloxy NCA (LPP(4R)-NCA) or L-proline-(4S)-propargyloxy NCA (LPP(4S)-NCA). NOESY and circular dichroism (CD) analysis indicate that incorporation of 4-substituted proline residues preserves the characteristic polyproline II (PPII) helical conformation, regardless of sequence architecture or substituent stereochemistry. Although all copolymers exhibit identical secondary structures at ambient temperature, statistical copolymers demonstrate significantly enhanced thermal stability and reduced aggregation compared to both the poly(L-proline) homopolymer and compositionally similar diblock copolymers. In contrast, diblock copolymers undergo thermally induced conformational transitions and aggregation similar to those observed in the PLP homopolymer, indicating that sequence distribution plays an important role in the thermal stability and aggregation propensity of these copolymers. Post-polymerization conjugation of solvatochromic fluorophores enables direct monitoring of sequence-dependent thermoresponsive assemblies, with fluorescence spectroscopy and microscopy supporting the variable-temperature CD and dynamic light scattering results. These findings establish sequence engineering as the dominant molecular design parameter for decoupling conformational stability from aggregation in polyproline-based materials and provide a versatile strategy for developing thermally robust and stimuli-responsive polypeptides.