The increasing prevalence of type 2 diabetes presents a major global health challenge. Glucagon-like peptide-1 (GLP-1) receptor agonists offer glycemic control, weight loss, and cardiovascular benefits but are limited by rapid enzymatic degradation and short half-life. Semaglutide, modified with a C18 diacid, extends its half-life to one week. Building on this, we developed GLP-1 analogues incorporating a hydrophilic proline-alanine-serine (PAS) sequence, referred to as PAS100, which comprises 100 amino acids with repetitive Pro, Ala, and Ser residues, along with dual fatty acid modifications, to evaluate their long-term therapeutic effects on glycemic control and body weight regulation. Fifteen conjugates were synthesized, featuring dual fatty acid modifications at Lysof Gly-Arg-GLP-1(7-37), with a PAS sequence at the C-terminus to mitigate lipidation-induced hydrophobicity. Seven exhibited enhanced GLP-1 receptor activation compared to their counterparts. Among them, B1, with two C14 monoacids and PAS100, exhibited approximately 3-fold increase in receptor activation compared to semaglutide. These seven conjugates were further evaluated in vivo, categorized into two groups based on their structural features, and compared with semaglutide for weight loss and blood glucose reduction in mice. Among these conjugates, compounds D1, D3, and D4 demonstrated glucose-lowering and weight-reducing effects comparable to semaglutide in vivo. Solubility measurements further confirmed that all d-series compounds displayed improved aqueous solubility, with D1 (bearing both a C14 monoacid and a C12 diacid and a PAS100) achieving the greatest enhancement, approximately two-fold higher than semaglutide. Surface plasmon resonance (SPR) analysis revealed that D1 exhibited a Kapproximately three-fold lower than that of semaglutide, indicating an enhanced receptor-binding tendency. In conclusion, D1 emerges as a promising GLP-1 analogue with significant therapeutic potential for the treatment of type 2 diabetes. Moreover, the dual-fatty acid conjugation strategy combined with PASylation represents a versatile platform that may be extended to other lipidated peptide therapeutics. 26 8 34D
