title: Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization.
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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Masayo
authors.initials: M
authors.lastname: Sakabe
authors.forename: Daisuke
authors.initials: D
authors.lastname: Asanuma
authors.forename: Mako
authors.initials: M
authors.lastname: Kamiya
authors.forename: Ryu J
authors.initials: RJ
authors.lastname: Iwatate
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
mesh_terms: D000818:Animals; D002417:Cattle; D003500:Cyclization; D010450:Endopeptidases; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D018093:Gelatinases / Q000032:analysis* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000032:analysis* / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D018613:Microscopy, Confocal; D015394:Molecular Structure; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013141:Spiro Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism; D000099167:Fibroblast Activation Protein Alpha
chemical_list: D010450:Endopeptidases; D005456:Fluorescent Dyes; D018093:Gelatinases; D007931:Leucyl Aminopeptidase; D008565:Membrane Proteins; D012235:Rhodamines; D012697:Serine Endopeptidases; D013141:Spiro Compounds; D001616:beta-Galactosidase; D000099167:Fibroblast Activation Protein Alpha