Two analogues of insulin glargine containing a 1,4-disubstituted 1,2,3-triazole group in place of the CysA7-CysB7 disulfide bond were prepared using CuAAC click chemistry to efficiently join the peptide chains. The resulting insulin analogues were analysed by circular dichroism spectroscopy to assess whether this modification compromised the folding pattern of the native form. Investigations, including an in-vivo murine study, revealed that these analogues were not biologically active and that the structures were significantly unfolded, an outcome which suggests that maintaining a precise inter-chain distance is critical to the structure of the insulin hormone.