1. Academic Validation
  2. Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization

Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization

  • J Am Chem Soc. 2013 Jan 9;135(1):409-14. doi: 10.1021/ja309688m.
Masayo Sakabe 1 Daisuke Asanuma Mako Kamiya Ryu J Iwatate Kenjiro Hanaoka Takuya Terai Tetsuo Nagano Yasuteru Urano
Affiliations

Affiliation

  • 1 Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Abstract

We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target Enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target Enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine Aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target Enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biological and pathological investigations.

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