A BODIPY-Tagged Phosphono Peptide as Activity-Based Probe for Human Leukocyte Elastase

Abstract

Human leukocyte elastase plays a crucial role in a variety of inflammatory disorders and represents an important subject of biomedical studies. The chemotype of peptidic phosphonates was employed for the design of a new activity-based probe for human leukocyte elastase. Its structure combines the phosphonate warhead with an adequate peptide portion and a BODIPY fluorophore with a clickable ethinylphenyl moiety at meso position. The probe 6 was assembled by copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition. It was characterized as an active site-directed elastase inhibitor exhibiting a second-order rate constant of inactivation of 88400 M^-1s^-1. The suitability of 6 as a fluorescent probe for human leukocyte elastase was demonstrated by in-gel fluorescence analysis. Labeling experiments and inhibition data toward a panel of related proteases underlined the selectivity of the probe for the targeted leukocyte elastase.

Scheme 1. Synthesis of the Activity-Based Probe 6

Reagents and conditions: (a) 50% TFA/CH₂Cl₂ (v/v), rt; (b) HBTU, DIPEA, MeCN, rt; (c) CuSO₄·5 H₂O, sodium ascorbate, H₂O, DMSO, rt.

Figure 1

Detection of HLE after labeling with the fluorescent probe 6 and SDS-PAGE. (A, C, E) In-gel fluorescence detection. (B, D, F) Coomassie staining. (A, B) Increasing amounts of HLE were incubated with 2.5 μM 6. (C, D) HEK cell lysate (9 μg, first lane) or HLE (600 ng, second lane) was incubated with 2.5 μM 6. HEK cell lysate (9 μg) was added after (third lane) or before (fourth lane) incubation of HLE (600 ng) with 2.5 μM 6. The horizontal line indicates the incubation period; the HEK cell lysate was added either before or after the incubation time. (E, F) HLE (600 ng) was incubated with or without 5 μM sivelestat (Siv) followed by incubation with 2.5 μM 6. M, molecular mass marker.