*Article* **Effect of C-terminus Conjugation via Different Conjugation Chemistries on In Vivo Activity of Albumin-Conjugated Recombinant GLP-1**

**Junyong Park 1,†, Mijeong Bak 2,†, Kiyoon Min 2,† , Hyun-Woo Kim <sup>3</sup> , Jeong-Haeng Cho <sup>3</sup> , Giyoong Tae <sup>2</sup> and Inchan Kwon 1,2,3,\***


**Abstract:** Glucagon-like peptide-1 (GLP-1) is a peptide hormone with tremendous therapeutic potential for treating type 2 diabetes mellitus. However, the short half-life of its native form is a significant drawback. We previously prolonged the plasma half-life of GLP-1 via site-specific conjugation of human serum albumin (HSA) at position 16 of recombinant GLP-1 using site-specific incorporation of p-azido-phenylalanine (AzF) and strain-promoted azide-alkyne cycloaddition (SPAAC). However, the resulting conjugate GLP1\_8G16AzF-HSA showed only moderate in vivo glucoselowering activity, probably due to perturbed interactions with GLP-1 receptor (GLP-1R) caused by the albumin-linker. To identify albumin-conjugated GLP-1 variants with enhanced in vivo glucoselowering activity, we investigated the conjugation of HSA to a C-terminal region of GLP-1 to reduce steric hindrance by the albumin-linker using two different conjugation chemistries. GLP-1 variants GLP1\_8G37AzF-HSA and GLP1\_8G37C-HSA were prepared using SPAAC and Michael addition, respectively. GLP1\_8G37C-HSA exhibited a higher glucose-lowering activity in vivo than GLP1\_8G16AzF-HSA, while GLP1\_8G37AzF-HSA did not. Another GLP-1 variant, GLP1\_8A37C-HSA, had a glycine to alanine mutation at position 8 and albumin at its C-terminus and exhibited in vivo glucose-lowering activity comparable to that of GLP1\_8G37C-HSA, despite a moderately shorter plasma half-life. These results showed that site-specific HSA conjugation to the C-terminus of GLP-1 via Michael addition could be used to generate GLP-1 variants with enhanced glucoselowering activity and prolonged plasma half-life in vivo.

**Keywords:** plasma half-life extension; albumin conjugation; in vivo glucose-lowering activity; glucagon-like peptide-1
