To generate thiol-reactive calcitonin analogue, synthetic salmon calcitonin (sCT, Calbiochem, USA) in DMSO (13.72 mg/ml) was mixed with NHS-PEG-MAL (Creative Biochem, USA) in DMSO (51 mg/ml) (Sigma,USA) in 1:3 molar ratio. DMSO was chosen as the reaction medium because of the instability of NHS and sCT in aqueous solutions. sCT is further highly soluble and highly stable in DMSO (20) . The reaction between the primary amines in sCT and NHS group of NHS-PEG-MAL was allowed to proceed at room temperature with constant stirring for 45 min. sCT has three primary amines at residues Lys 11, Lys 18 and the N-terminus, which can react with the NHS functional group of NHS-PEG-MAL to generate three intermediate conjugates: mono-, di-and tri-substituted thiol reactive sCT analogues (Fig. 1b) . 