New Biarsenical Derivatives for specific labeling

CARLA C. SPAGNUOLO; RUDOLPH J. VERMEIJ; ELIZABETH JARES-ERIJMAN

Salvador, Bahía, Brasil

Congreso; 17th Inter-American Photochemical Society Winter Conference.; 2006

Inter-American Photochemical Society

The field of molecular physiology aims at the elucidation of the (bio)chemical and (bio)physical molecular mechanisms underlying the hierarchical and tightly networked flow of matter, and energy,characteristic of biological cells and organisms. The development of this field is strongly dependent on the availability of a wide repertoire of expression probes. These consist of peptides, proteins or nucleic acids that can be expressed on the surface or within cells, and which display an intrinsic and specific fluorescent signal or constitute a target for a fluorescent probe supplied externally and taken up by the cell. Despite their versatility, the widely used visible fluorescent proteins (VFPs) are unsuitable for many applications due to their large size (27 KDa) and limited photostability, particularly for imaging at the single molecule level. Small-molecule probes are thus desirable in that they offer reduced stereochemical interference, faster rates of labeling, photochemical stability, and the ability to provide readouts in addition to, or other than, fluorescence. Among the methodologies that combine exogenous small molecule probes with short peptide tags, biarsenical fluorescein derivatives such as FlAsH, are being increasingly applied. FlAsH is a membrane-permeable fluorogenic dye, which form highly stable complexes with 6-12-mer peptide tags, containing a CCXXCC motif, that are engineered in the protein of interest. However, the poor photostability and pH sensitivity of fluorescein derivatives in the fisiological range constitute major limitations which preclude a more widespread application of the method. In order to overcome these limitations we have synthesized fluorinated FlAsH derivatives F2FlAsH, F4FlAsH (Figure). Upon complex formation with the 12-mer FLNCCPGCCMEP the novel biarsenical probes there is a large increase (up to four-fold) in fluorescence emission intensity in comparison to the parent FlAsH probes. In addition, F2FlAsH complexes display a substantially greater photostability than FlAsH complexes. Both characteristics make these new probes superior to FlAsH for live cell microscopy.