title: Synthesis, in vitro evaluation, and in vivo metabolism of fluor/quencher compounds containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3).
abstract: Protease-cleavable peptides containing a suitable fluor/quencher (Fl/Q) pair are optically dark until cleaved by their target protease, generating fluorescence. This approach has been used with many Fl/Q pairs, but little has been reported with IRDye 800CW, a popular near-infrared (NIR) fluor. We explored the use of the azo-bond-containing Black Hole Quencher 3 (BHQ-3) as a quencher for IRDye 800CW and found that IRDye 800CW/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Cleavage of IRDye 800CW-PLGLK(BHQ-3)AR-NH(2) (8) and its D-arginine (Darg) analogue (9) by matrix metalloproteinases (MMPs) in vitro yielded the expected cleavage fragments. In vivo, extensive metabolism was found. Significant decomposition of a "non-cleavable" control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane)-BHQ-3 (10) was evident in plasma of normal mice by 3 min post injection. The major metabolite showed a m/z and UV/vis spectrum consistent with azo bond cleavage in the BHQ-3 moiety. Preparation of an authentic standard of this metabolite (11) confirmed the assignment. Although the IRDye 800CW/BHQ-3 constructs showed efficient contact quenching prior to enzymatic cleavage, BHQ-3 should be used with caution in vivo, due to instability of its azo bond.
authors.affiliation: Ernst Felder Laboratories, Bracco Research USA, 305 College Road East, Princeton, New Jersey 08540, United States. karen.e.linder@gmail.com
authors.forename: Karen E
authors.initials: KE
authors.lastname: Linder
authors.forename: Edmund
authors.initials: E
authors.lastname: Metcalfe
authors.forename: Palaniappa
authors.initials: P
authors.lastname: Nanjappan
authors.forename: Thangavel
authors.initials: T
authors.lastname: Arunachalam
authors.forename: Kimberly
authors.initials: K
authors.lastname: Ramos
authors.forename: Tina Marie
authors.initials: TM
authors.lastname: Skedzielewski
authors.forename: Edmund R
authors.initials: ER
authors.lastname: Marinelli
authors.forename: Michael F
authors.initials: MF
authors.lastname: Tweedle
authors.forename: Adrian D
authors.initials: AD
authors.lastname: Nunn
authors.forename: Rolf E
authors.initials: RE
authors.lastname: Swenson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001557:Benzenesulfonates / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D007211:Indoles / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D008297:Male; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D001557:Benzenesulfonates; D005456:Fluorescent Dyes; C562366:IRDye 800CW; D007211:Indoles; D010455:Peptides; D020782:Matrix Metalloproteinases