AMIDRAZONE ANALOGS OF D-RIBOFURANOSE AS TRANSITION-STATE INHIBITORS OF NUCLEOSIDE HYDROLASE

The transition state of inosine during hydrolysis by nucleoside hydrol ase has been characterized by kinetic isotope effects, bond-energy/bon d-order vibrational analysis, and molecular electrostatic potential su rface calculations [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795; Horenstein, B. A. , & Schramm, V. L. (1993) Biochemistry 32, 7089-7097]. The heterocycli c base is protonated and the anomeric carbon of the ribofuranosyl ring is flattened to form a transition-state with extensive oxocarbenium i on character. With their delocalized charge and flattened structures, amidrazone analogues of D-ribofuranose provide both geometric and elec tronic mimics of the ribosyl group at the transition-state of nucleosi de hydrolase. A family of riboamidrazones was synthesized with H, phen yl, and p-nitrophenyl N-substituents. The analogues were competitive i nhibitors with respect to inosine and gave K(i) values of 10(-5), 2 x 10(-7), and 1 x 10(-8) M, respectively. (p-Nitrophenyl)riboamidrazone exhibited slow-onset, tight-binding inhibition, with an overall dissoc iation constant of 2 x 10(-9) M. The binding is reversible with an off -rate of 3 x 10(-3) S-1. Tight binding can be attributed to the close spatial match between the molecular geometry of (p-nitrophenyl)riboami drazone and the transition-state stabilized by nucleoside hydrolase. T he favorable binding interactions of the (p-nitrophenyl)riboamidrazone include oxocarbenium ion mimicry, isosteric ribosyl hydroxyls, and hy drophobic and H-bonding interactions at the nitrophenyl group. Analysi s of the conformational space available to the (p-nitrophenyl)riboamid razone indicates that the geometry that approximates the enzyme-stabil ized transition state is 7-14 kcal/mol unfavorable relative to the glo bal conformational minimum for free inhibitor. The apparent overall K( d) of 2 nM represents only a fraction of the intrinsic energy availabl e for transition-state interactions with nucleoside hydrolase. When co rrected for the energy of distortion required to achieve the transitio n-state conformation, (p-nitrophenyl)riboamidrazone binds with an affi nity near that expected for an ideal transition-state analogue.