This study aimed to develop and optimize chemistries to produce alkyne-modified glucagon-like peptide-1(7−36)-amide (GLP-1(7−36)-NH 2 ) libraries, which could be rapidly and efficiently conjugated to other components and screened to identify compounds with the best drug delivery properties, as potential treatments for type 2 diabetes or obesity. For this purpose, the Lys26 (K26) side-chain, and the amino (N)and carboxy (C)-termini of a dipeptidyl peptidase 4 (DPPIV)resistant GLP-1 sequence (GLP-1(7−36;A8G)-NH 2 ), were modified with an alkyne (4-pentynoic acid or propiolic acid). These analogs were characterized with respect to human GLP-1 receptor (hGLP-1R) agonist activity, effects on cell viability and human serum stability, revealing that these modifications maintained low (N-terminal; EC 50 1.5 × 10 −9 M) to subnanomolar (C-terminal and K26, ∼4 × 10 −10 M) agonist activity toward hGLP-1, had no effect on cell viability, and for the N-terminal and K26 modifications, increased human serum proteolytic stability (t 1/2 > 24 h). Copper-catalyzed azide−alkyne cycloaddition (CuAAC) reaction conditions were investigated using the Cterminal modified GLP-1 analog and an azide-modified model lipid peptide, with respect to the effects of altering the azide/alkyne ratio, cosolvents, temperature, reducing agents, Cu(I)-stabilizing ligand, copper source, and the concentrations of reagents/reactants, in order to identify general conditions that provide fast reactions and high yields. A 1:2 azide−alkyne (lipid:GLP-1 peptide) and 4:1 sodium ascorbate/copper sulfate molar ratio in 65% v/v DMSO−water at room temperature, in the absence of Cu(I)-stabilizing ligands (THPTA or L-histidine) and buffers (phosphate, pH 7), provided the best yields. This work reports a library of characterized GLP-1 analogs and chemistries for their attachment to other species, providing useful tools to improve GLP-1 delivery and pharmacology (e.g., through conjugation to other species that lower blood glucose, increase the duration of action, or enable delivery via a nonparenteral route).
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