Peptide targeting of fluorescein-based sensors to discrete intracellular locales

Abstract

Fluorescein-based sensors are the most widely applied class of zinc probes but display adventitious localization in live cells. We present here a peptide-based localization strategy that affords precision in targeting of fluorescein-based zinc sensors. By appending the zinc-selective, reaction-based probe Zinpyr-1 diacetate (DA-ZP1) to the N-terminus of two different targeting peptides we achieve programmable localization and avoid unwanted sequestration within acidic vesicles. Furthermore, this approach can be generalized to other fluorescein-based sensors. When appended to a mitochondrial targeting peptide, the esterase-activated profluorophore 2′,7′-dichlorofluorescein diacetate can be used effectively at concentrations four-times lower than previously reported for analogous, non-acetylated derivatives. These results demonstrate on-resin or in-solution esterification of fluorescein to be an effective strategy to facilitate peptide-based targeting in live cells.

Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues

“…First, we investigated a diacetylated (DA) version of the fluorescein-based sensor, ZP1, in the absence of a targeting vector in order to directly characterize the effects of acetylation on zinc binding, fluorescence response, and cellular localization. 13,14 DA-ZP1 is spectroscopically silent at wavelengths >400 nm and nonfluorescent. Zinc binding promotes cleavage of the acetyl groups, restoring visible absorption (λ max = 505 nm) and emission (λ em = 525 nm, Φ = 0.77), features consistent with those reported for zinc-bound ZP1 and DA-ZP1 conjugates.…”

“…24 When EDTA is introduced to remove zinc from the sensor, absorption and emission features identical to those reported for zinc-free ZP1 appear (λ abs = 515 nm, λ em = 531 nm), consistent with removal of the acetyl groups. 13,14 Next, we investigated whether acetylation of PET-based sensors with other fluorophores also quenches background fluorescence. Previously, a DPA unit was appended onto 7-hydroxycoumarin to yield a blue zinc sensor, CM1.…”

“…Previously, we showed that diacetylation (DA) of Zinpyr1 (ZP1) appended to (6-amidoethyl)triphenylphosphonium (TPP) 13 or various targeting peptides 14 effectively quenches background fluorescence by disrupting the π-conjugation of fluorescein, resulting in a substantially increased zinc-induced fluorescence response by avoiding proton-induced turn-on. In the case of DA-ZP1 derivatives, the zinc ion performs two functions: (i) its Lewis acidity promotes rapid hydrolysis of the ester groups, restoring conjugation of the fluorophore, and (ii) coordination to the two dipicolylamine (DPA) zinc-binding units attenuates PET.…”

Green and Red Fluorescent Dyes for Translational Applications in Imaging and Sensing Analytes: A Dual‐Color Flag

“…A shortcoming of these probes is that they suffer from unpredictable cellular localization. In an attempt to design a more accurate delivery system, Radford and co‐workers have published a peptide‐based targeting strategy, in which a series of targeting peptides are attached to the diacetylated Zinpyr sensor 15 for intracellular detection of Zn II (see Figure ). Modification of probe 15 with a mitochondrial‐targeting peptide allows the use of the sensor in concentrations four times lower than previously reported for the non‐acetylated probe.…”

Bioorthogonal Turn‐On BODIPY‐Peptide Photosensitizers for Tailored Photodynamic Therapy

“…[21] To investigate our hypothesis, we aimed for the synthesis and study of five bioorthogonally aPS peptidec onjugates ( Figure 1, Tz-C(2I-BODIPY)-PEPTIDE, 2-6OFF)a nd the corresponding active PS analogues: [TCO:Tz]-C(2I-BODIPY)-PEPTIDE, 2-6ON.W es elected the conventional polyarginines (Rn) composed of five (R5) (Tz-C(2I-BODIPY)-R5, 2OFF and [TCO:Tz]-C(2I-BODIPY)-R5, 2ON)o reight (R8) (Tz-C(2I-BODIPY)-R8, 3OFF and [TCO:Tz]-C(2I-BODIPY)-R8, 3ON)c onsecutive arginine residues capable of delivering cargos to the cytoplasm and/ornucleus, [22] as well as two mitochondrial-penetrating peptides( MPPs)f rom Kelley's lab (Tz-C(2I-BODIPY)-MPP1, 4OFF and [TCO:Tz]-C(2I-BODIPY)-MPP1, 4ON;T z-C(2I-BODIPY)-MPP2, 5OFF and [TCO:Tz]-C(2I-BODIPY)-MPP2, 5ON), as our targeting vectors. Thel atterp eptides, MPP1 [23] and MPP2, [24] are based on the combination of two unnatural amino acids:c yclohexylalanine (F x )a nd d-arginine (r), the difference between them is only as ingle extra d-arginine residue in MPP2. We also pursued the preparation of a conjugate pair embedded in the cell membrane.…”