Spiropyran-Based Photoisomerizable α-Amino Acid for Membrane-Active Peptide Modification.

Photoisomerizable peptides are promising drug candidates in photopharmacology. While azobenzene- and diarylethene-containing photoisomerizable peptides have already demonstrated their potential in this regard, reports on the use of spiropyrans to photoregulate bioactive peptides are still scarce. This work focuses on the design and synthesis of a spiropyran-derived amino acid, (S)-2-amino-3-(6'-methoxy-1',3',3'-trimethylspiro-[2H-1-benzopyran-2,2'-indolin-6-yl])propanoic acid, which is suitable for the preparation of photoisomerizable peptides. The utility of this amino acid is demonstrated by incorporating it into the backbone of BP100, a known membrane-active peptide, and by examining the photoregulation of the membrane perturbation by the spiropyran-containing peptides. The toxicity of the peptides (against the plant cell line BY-2), their bacteriotoxicity (E. coli), and actin-auxin oscillator modulation ability were shown to be significantly dependent on the photoisomeric state of the spiropyran unit.

Highly Fluorinated Peptide Probes with Enhanced In Vivo Stability for 19 F-MRI.

A labeling strategy for in vivo 19 F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and ß-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 19 F substitutions chemically equivalent, giving rise to a single 19 F-NMR resonance with <10 Hz linewidth. The resulting performance in 19 F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in 19 F-NMR linewidth to ≈30 Hz. Overall, these peptide probes are hence suitable for in vivo 19 F-MRI applications.

In Vivo Behavior of the Antibacterial Peptide Cyclo[RRRWFW], Explored Using a 3-Hydroxychromone-Derived Fluorescent Amino Acid.

Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates in vivo.

Diarylethene-Based Photoswitchable Inhibitors of Serine Proteases.

A bicyclic peptide scaffold was chemically adapted to generate diarylethene-based photoswitchable inhibitors of serine protease Bos taurus trypsin 1 (T1). Starting from a prototype molecule-sunflower trypsin inhibitor-1 (SFTI-1)-we obtained light-controllable inhibitors of T1 with Ki in the low nanomolar range, whose activity could be modulated over 20-fold by irradiation. The inhibitory potency as well as resistance to proteolytic degradation were systematically studied on a series of 17 SFTI-1 analogues. The hydrogen bond network that stabilizes the structure of inhibitors and possibly the enzyme-inhibitor binding dynamics were affected by isomerization of the photoswitch. The feasibility of manipulating enzyme activity in time and space was demonstrated by controlled digestion of gelatin-based hydrogel and an antimicrobial peptide BP100-RW. Finally, our design principles of diarylethene photoswitches are shown to apply also for the development of other serine protease inhibitors.

Nature of Fast Relaxation Processes and Spectroscopy of a Membrane-Active Peptide Modified with Fluorescent Amino Acid Exhibiting Excited State Intramolecular Proton Transfer and Efficient Stimulated Emission.

A fluorescently labeled peptide that exhibited fast excited state intramolecular proton transfer (ESIPT) was synthesized, and the nature of its electronic properties was comprehensively investigated, including linear photophysical and photochemical characterization, specific relaxation processes in the excited state, and its stimulated emission ability. The steady-state absorption, fluorescence, and excitation anisotropy spectra, along with fluorescence lifetimes and emission quantum yields, were obtained in liquid media and analyzed based on density functional theory quantum-chemical calculations. The nature of ESIPT processes of the peptide's chromophore moiety was explored using a femtosecond transient absorption pump-probe technique, revealing relatively fast ESIPT velocity (∼10 ps) in protic MeOH at room temperature. Efficient superluminescence properties of the peptide were realized upon femtosecond excitation in the main long-wavelength absorption band with a corresponding threshold of the pump pulse energy of ∼1.5 µJ. Quantum-chemical analysis of the electronic structure of the peptide was performed using the density functional theory/time-dependent density functional theory level of theory, affording good agreement with experimental data.