AbstractThe glycosylation of neuroactive peptides is a promising strategy to treat neurological and psychiatric disorders. Herein we investigated the effects of site‐specific glycosylation of neurotensin (NT). The glycosylated analogues have low‐nanomolar affinities and agonist activities toward NTS1, and suppress seizures with sub‐picomolar potency. Our work points to a new research direction of exploring BBB‐permeable NT analogues as potential first‐in‐class antiepileptic drugs.magnified imageNeurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Herein we show the effects of site‐specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogues containing O‐linked disaccharides (β‐melibiose and α‐TF antigen) or β‐lactose units linked by a PEG3 spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analogue, Fmoc‐Glu‐(β‐Lac‐PEG3‐amide) was prepared. Our results indicate that the addition of the disaccharides does not negatively affect the sub‐nanomolar affinity or the low‐nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogues exhibited sub‐picomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically modified NT analogues may lead to novel antiepileptic therapies.