Synthesis, in vitro evaluation, and in vivo metabolism of fluor/quencher compounds containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3)

Bioconjug Chem. 2011 Jul 20;22(7):1287-97. doi: 10.1021/bc100457s. Epub 2011 Jun 27.

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Benzenesulfonates / chemical synthesis
  • Benzenesulfonates / chemistry*
  • Benzenesulfonates / metabolism*
  • Benzenesulfonates / pharmacokinetics
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism*
  • Fluorescent Dyes / pharmacokinetics
  • Indoles / chemical synthesis
  • Indoles / chemistry*
  • Indoles / metabolism*
  • Indoles / pharmacokinetics
  • Male
  • Matrix Metalloproteinases / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Peptides / chemical synthesis
  • Peptides / chemistry*
  • Peptides / metabolism*
  • Peptides / pharmacokinetics

Substances

  • Benzenesulfonates
  • Fluorescent Dyes
  • IRDye 800CW
  • Indoles
  • Peptides
  • Matrix Metalloproteinases