Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis

Introduction

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Fluorescent peptides are essential tools used extensively in drug discovery and disease diagnosis. Although EDANS, FAM, and TAMRA are widely used to label peptides, their short absorption wavelengths, low extinction coefficients, and pH-sensitive fluorescence have limited their use in developing sensitive fluorescent peptide probes. Alexa Fluor^® (AF) dyes (Invitrogen) demonstrated improved fluorescence properties. However, the high cost of Alexa Fluors makes the AF dyes unaffordable for labeling peptides.

The strongly fluorescent and affordable Tide Fluor™ (TF) dyes eliminate the limitations mentioned above of the classic dyes. They are individually optimized for labeling peptides and nucleotides. As the Alexa Fluors, the TF dyes are bright, pH-insensitive, and photostable. Compared to Alexa Fluors, TF dyes are more stable to the peptide deprotection conditions.

We have used TF 2 and TF5 dyes to develop new fluorescent peptides for high throughput analysis of protease and protein kinase activities and screening of protease and protein kinase inhibitors. Excellent spectral properties and labeling efficiencies have also been observed for TF3, 4, 6, 7, and 8. Among them, TF 6, 7, and 8 have been used for developing fluorescent peptides that have emissions in the near-infrared or infrared range. These NIR and IR TF-based peptides have been successfully used in in-vivo imaging.

Materials and Method

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All the peptides were synthesized using the standard FMOC chemistry by the American Peptide Company. Some of the TF peptides were prepared using FMOC-Lys(TF)-OH, FMOCAsp(TF)-OH, or FMOC-Glu(TF)-OH amino acids. For the post-labeling of the premade peptides, TF NHS esters were used for labeling the N-terminal amino or ε–amino group of a lysine residue. TF maleimides or iodoacetamides were used for labeling the SH group of cysteine residues.

TF1, TF2, TF3, TF4, TF5, TF6, TF7, and TF8 acids, maleimides and NHS esters, FMOC-Lys(TF2-Boc)-OH, FMOCLys(TF3)-OH, FMOC-Asp(TF2-Boc)-OH, FMOC-Asp(TF3)-OH, FMOC-Glu(TF3)-OH, FMOCGlu(TF2-Boc)-OH and FMOC-Glu(TF3)-OH are now commercially available from AAT Bioquest.

All the enzyme assays were done with purified enzymes commercially available from Sigma, R&D Systems, EMD Chemicals. Absorption spectra were taken with Hitachi U-3010. Endpoint fluorescence assays were run on either BMG NovoStar or Gemini SpectraMax (Molecular Devices). Enzyme kinetic assays were run on FlexStation Molecular Devices).

Spectral Properties of Tide Fluor™ (TF) Dyes

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Our TF series of fluorescent labeling dyes cover the entire visible spectrum with unusually high labeling efficiency. For example, TF2 has spectral properties essentially identical to AF 488, FAM, and FITC. It is three times brighter and four times more photostable than Fluorescein under physiological conditions or in cells. TF dyes have fluorescence insensitive to pH fluctuation from pH 4 to pH 10, while the fluorescence intensity of FAM and FITC is strongly dependent on pH in the same range.

1. Legends: AF = Alexa Fluor^® (Invitrogen); DL = DyLight™ (Thermo Fisher); IRDye CW800 (Li-Cor); TF = Tide Fluor™ (AAT Bioquest)

Detection of Matrix Metalloproteinase (MMP) Activities Using TF-Based FRET Substrates

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Matrix metalloproteinases (MMPs) are involved in the degradation of components of the extracellular matrix and play an important role in apoptosis, embryogenesis, reproduction, tissue remodeling, and repair. The following TF-based FRET substrates were synthesized and screened for detecting MMP activities.

Detection of Generic Protease Activities Using TF-Labeled Casein Substrates

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Monitoring of various protease activities has become a routine task for many biological laboratories. TF dyes-labeled casein conjugates are proven to be generic substrates for a broad spectrum of proteases. In the intact substrates, casein is heavily labeled with a TF dye, resulting in significant fluorescence quenching. Protease-catalyzed hydrolysis relieves its quenching effect, yielding brightly fluorescent TF dye-labeled peptide fragments. The increase in fluorescence intensity of TF dye-labeled peptide is directly proportional to the protease activity.