Published on Web 07/01/2009
A Bifunctional Molecule as an Artificial Flavin Mononucleotide
Cyclase and a Chemosensor for Selective Fluorescent
Detection of Flavins
Hyun-Woo Rhee,† So Jung Choi,† Sang Ho Yoo,† Yong Oh Jang,† Hun Hee Park,‡
Rosa Mar´ıa Pinto,§ Jose´ Carlos Cameselle,§ Francisco J. Sandoval,| Sanja Roje,|
Kyungja Han,‡ Doo Soo Chung,† Junghun Suh,*,† and Jong-In Hong*,†
Department of Chemistry, College of Natural Sciences, Seoul National UniVersity, Seoul
151-747, Korea, Department of Clinical Pathology, Catholic UniVersity Medical College, Seoul,
Korea, Grupo de Enzimolog´ıa, Departamento de Bioqu´ımica y Biolog´ıa Molecular y Gene´tica,
Facultad de Medicina, UniVersidad de Extremadura, 06080 Badajoz, Spain, and Institute of
Biological Chemistry, Washington State UniVersity, Pullman, Washington 99164
Received March 8, 2009; E-mail: jihong@snu.ac.kr; jhsuh@snu.ac.kr
Abstract: Flavins, comprising flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin
(RF, vitamin B2), play important roles in numerous redox reactions such as those taking place in the electron-
transfer chains of mitochondria in all eukaryotes and of plastids in plants. A selective chemosensor for
flavins would be useful not only in the investigation of metabolic processes but also in the diagnosis of
diseases related to flavins; such a sensor is presently unavailable. Herein, we report the first bifunctional
chemosensor (PTZ-DPA) for flavins. PTZ-DPA consists of bis(Zn2+-dipicolylamine) and phenothiazine.
Bis(Zn2+-dipicolylamine) (referred to here as XyDPA) was found to be an excellent catalyst in the conversion
of FAD into cyclic FMN (riboflavin 4′,5′-cyclic phosphate, cFMN) under physiological conditions, even at
pH 7.4 and 27 °C, with less than 1 mol % of substrate. Utilizing XyDPA’s superior function as an artificial
FMN cyclase and phenothiazine as an electron donor able to quench the fluorescence of an isoalloxazine
ring, PTZ-DPA enabled selective fluorescent discrimination of flavins (FMN, FAD, and RF): FAD shows
ON(+), FMN shows OFF(-), and RF shows NO(0) fluorescence changes upon the addition of PTZ-DPA.
With this selective sensing property, PTZ-DPA is applicable to real-time fluorescent monitoring of riboflavin
kinase (RF to FMN), alkaline phosphatase (FMN to RF), and FAD synthetase (FMN to FAD).
Introduction
However, many artificial metalloenzymes operate efficiently
only at high temperatures over 50 °C, requiring a relatively large
Development of an artificial enzyme with activity comparable
to that of natural enzymes is an important goal in chemical
biology.1-3 Artificial metalloenzymes2,3 have been developed
to mimic the catalytic metallic core of natural enzymes.
amount of enzyme compared to that of substrate such as nucleic
acids,2e,3a proteins,2b or dinucleotides.2f,3c,e Reports of artificial
enzymes working promptly in physiological conditions with less
than 1 mol % of a substrate are rare.
Recently, fluorescent chemosensors have been intensively
developed to detect specific biomolecules or monitor biological
† Seoul National University.
‡ Catholic University Medical College.
events.4,5 In particular, many chemosensors based on bis(Zn2+
-
§ Universidad de Extremadura.
| Washington State University.
DPA) (DPA ) dipicolylamine) complex5 have been synthesized
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10.1021/ja9018012 CCC: $40.75 2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 10107–10112 10107