The Effect of Protecting Groups and Peptide Chain Length on the Aqueous Solubility of l‑Alanine Homopeptides.

The solubility of peptides is a critical physicochemical parameter governing both upstream solid-phase peptide synthesis (SPPS) route optimization and downstream crystallization design. In this study, the aqueous solubility of l-alanine and its homologous oligopeptides bearing Boc and Cbz N-terminal protecting groups was systematically determined from 278.15 K to 313.15 K using the gravimetric method. N-capped peptide fragments exhibited aqueous solubility 90% lower than their unprotected counterparts, demonstrating that hydrophobic protecting groups substantially suppress aqueous dissolution. Notably, dialanine exhibited a 1.72-fold solubility enhancement relative to alanine at 298.15 K, and retrograde solubility behavior was observed for the first time for trialanine and tetraalanine. These anomalous findings were rigorously interpreted through analyses of solid-state crystal packing, thermodynamic properties, and molecular interactions. Experimental solubility data were correlated using the modified Apelblat equation. Complementary molecular dynamics simulations were employed to elucidate the intramolecular hydrogen-bonding networks. This work presents the first comprehensive solubility dataset for N-protected peptide fragments, offering physicochemical insights to inform protecting group selection and the development of downstream crystallization strategies.