Synthesis of Novel Fluorescent ATP Analogue and its Interaction with Nucleotide Dependent Motor Proteins

Abstract

Previously several kind of fluorescent ATP analogues have been synthesized for the application to the kinetic studies of ATPases. However, some of the ATP analogues exist as a mixture of isomers. For instance, 2′(3′)-O-Mant-ATP has isomer of 2′ and 3′ in its ribose moiety and each isomer performs differently as substrate for the ATPases. In the present study, we have tried to synthesize novel fluorescent ATP analogues that have no isomer. The fluorescent ATP analogue 6-(N- (7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino) ethyl triphosphate (NBDTP) and N-methylanthraniloyl amino ethyl triphosphate (MANTTP) have been designed and synthesized, which are similar to non-nucleotide ATP analogue 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP). It is known that NANTP are good substrate for skeletal myosin and induces actin gliding in vitro motility assay. Excitation and emission maximums in the fluorescence spectrum of the ATP analogues were 474nm and 533nm for NBDTP, and 374nm and 430nm for MANTTP, respectively. In addition, molar absorbance coefficients of ATP analogues were 40274 M−1cm−1 for NBDTP in 478nm, and 3730 M−1cm−1 for MANTTP, respectively. The analogues showed ATP hydrolysis for conventional kinesin and skeletal myosin at the almost same level to that of ATP. Moreover, the analogues induced dissociation of acto-myosin. The ADP form of the fluorescent ATP analogues showed the formation of skeletal muscle myosin/ADP analogues/BeFn complexes which mimic the transient state in ATPase cycle.