A New Fluorogenic Substrate for Granzyme B Based on Fluorescence Resonance Energy Transfer

The synthesis and characterization of a new fluorogenic substrate for granzyme B (GzmB) is reported. The substrate design was based on the fluorescence resonance energy transfer (FRET) principle using 5-(2′-aminoethyl)aminonaphthalene sulfonic acid (Edans) and 4-[[4′-(N,N-dimethylamino)phenyl]diazenyl]benzoic acid (Dabcyl) as a donor–acceptor pair, linked to a specific sequence for GzmB (AAD), with an additional amino acid as the anchoring point (K). The tetrapeptide was synthesized by microwave-assisted solid-phase peptide synthesis (MW-SPPS) and coupled to Dabcyl and Edans at its N- and C-termini, respectively. The obtained probe was purified by semi-preparative HPLC and characterized by NMR, UV/Vis absorption and fluorescence spectroscopy and mass spectrometry.

[1]  M. Raposo,et al.  Intermolecular Quenching of Edans/Dabcyl Donor–Acceptor FRET Pair , 2019, Proceedings.

[2]  K. Flaherty,et al.  Granzyme B PET Imaging as a Predictive Biomarker of Immunotherapy Response. , 2017, Cancer research.

[3]  N. Perkas,et al.  Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation , 2016, Applied Microbiology and Biotechnology.

[4]  R. Raines,et al.  Fluorogenic Assay for Inhibitors of HIV-1 Protease with Sub-picomolar Affinity , 2015, Scientific Reports.

[5]  J. Trapani,et al.  A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl. , 2014, Journal of visualized experiments : JoVE.

[6]  Ella F Jones,et al.  Activatable Optical Probes for the Detection of Enzymes. , 2011, Current organic synthesis.

[7]  P. Richardson,et al.  Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease. , 2007, Analytical biochemistry.

[8]  Katsumi Eguchi,et al.  Granzyme B and natural killer (NK) cell death , 2005, Modern rheumatology.

[9]  Fei Ye,et al.  Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique , 2005, Acta Pharmacologica Sinica.

[10]  R. M. Cook,et al.  Intramolecular dimers: a new design strategy for fluorescence-quenched probes. , 2003, Chemistry.

[11]  R. Sékaly,et al.  HIV-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation , 2002, Cell Death and Differentiation.

[12]  R. Huber,et al.  Crystal Structure of the Caspase Activator Human Granzyme B, a Proteinase Highly Specific for an Asp-P1 Residue , 2000, Biological chemistry.

[13]  Robert Fletterick,et al.  The structure of the pro-apoptotic protease granzyme B reveals the molecular determinants of its specificity , 2000, Nature Structural Biology.

[14]  D. Ullmann,et al.  Design and synthesis of fluorogenic trypsin peptide substrates based on resonance energy transfer. , 1998, Analytical biochemistry.

[15]  Jennifer L. Harris,et al.  Definition and Redesign of the Extended Substrate Specificity of Granzyme B* , 1998, The Journal of Biological Chemistry.

[16]  B. Dunn,et al.  A continuous fluorescence-based assay of human cytomegalovirus protease using a peptide substrate. , 1995, Analytical biochemistry.

[17]  A. Berger,et al.  On the size of the active site in proteases. I. Papain. , 1967, Biochemical and biophysical research communications.

[18]  Gary T. Wang,et al.  DESIGN AND SYNTHESIS OF NEW FLUOROGENIC HIV PROTEASE SUBSTRATES BASED ON RESONANCE ENERGY TRANSFER , 1990 .