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

Masayo Sakabe, Daisuke Asanuma, Mako Kamiya, Ryu J. Iwatate, Kenjiro Hanaoka, Takuya Terai, Tetsuo Nagano, Yasuteru Urano

Research output: Contribution to journalArticlepeer-review

203 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)409-414
Number of pages6
JournalJournal of the American Chemical Society
Issue number1
Publication statusPublished - 2013 Jan 9
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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