MARCH/APRIL 2006 Volume 17, Number 2 © Copyright 2006 by the American Chemical Society
COMMUNICATIONS Fluorescence Probe with a pH-Sensitive Trigger Amit K. Galande, Ralph Weissleder, and Ching-Hsuan Tung* Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129. Received November 17, 2005; Revised Manuscript Received February 7, 2006
Acid-catalyzed hydrolysis was used as the mechanism to design a new type of environmentally sensitive fluorescence probe. A mild and selective periodate oxidation of the 2-amino alcohol of serine in the presence of a disulfide bond was developed to prepare dialdehyde peptides. Two identical fluorochrome hydrazide derivatives were then linked to the dialdehyde peptide forming an acid-labile hydrazone linkage. This self-quenched probe is weakly fluorescent at a physiological pH of 7.4 but shows more than 3-fold fluorescence enhancement at pH 4.5.
Molecular probes for imaging applications are often designed using fluorescence resonance energy transfer (FRET)-related mechanisms to reduce background (1). FRET-based probes typically incorporate a pair of two different dye molecules, a fluorescent donor, and a fluorescent acceptor or a fluorochrome and a nonfluorescent quencher. Recently, self-quenched fluorescent probes consisting of multiple copies of the same fluorescent dye molecule have also been developed for molecular imaging (2, 3). Biological processes such as conformational changes of protein, receptor binding of ligands, and catalytic function of proteases have been exploited as molecular targets for various fluorescence emission probes (4-8). In addition, activity-based probes have also been developed for different enzyme classes (9, 10). In this report, a new approach involving acid-catalyzed hydrolysis as the mechanism of fluorescence activation is demonstrated. CY5G2, an aminopeptidase N (APN)-targeted fluorescence probe (Scheme 1), was constructed using an acid-sensitive hydrazone linkage. CY5G2 has been designed in such a way * To whom correspondence should be addressed: Ching H. Tung, Center for Molecular Imaging Research, Massachusetts General Hospital. 149 13th St., Rm. 5410, Charlestown, MA 02129. Tel: 617 726-5779, Fax: 617 726-5708 E-mail:
[email protected].
that it stays weakly fluorescent at the extracellular pH of 7.4 and becomes strongly fluorescent at the intracellular lysosomal pH of 4.5. CY5G2 incorporates a disulfide-constrained NGR (Asn-Gly-Arg) motif, which is a ligand for the APN receptor and is likely to facilitate entry of the probe into endosome and then into acidic lysosome (8, 11, 12). Here we report design and synthesis of CY5G2 along with its evaluation under physiologically relevant pH environments. The probe was constructed on a flexible polyglycine backbone. The N-terminus of the peptide was extended to incorporate the disulfide-bridged NGR motif, while toward the C-terminus two molecules of Cy5 were conjugated via an acid-sensitive hydrazone linkage. Cy5 dyes were chosen because of their farred fluorescence emission (670 nm). Fluorescence in the farred to near-infrared region (650-900 nm) has been found to be most suitable for in vivo optical imaging (13). The two Cy5 dyes on CY5G2 are separated from the disulfide-constrained NGR motif by two glycine residues. Another short spacer (
