Distorting Duplex DNA by Dimethylenesulfone Substitution:  A New Class of “Transition State Analog” Inhibitors for Restriction Enzymes

After they bind (but before they cleave) duplex DNA, some restriction enzymes (such as EcoRV and EcoRI) distort the duplex. The distorted duplex is not, of course, in its ground-state conformation; it requires "binding energy" to bend DNA. Thus, an analogue of DNA that generates this distortion in the unbound state (without altering other features of the substrate that are recognized by the enzyme) should bind to these restriction enzymes with a higher affinity than the DNA substrate itself. This is, of course, the principle underlying transition-state analogues generally, which approximate in structure the "distorted" transition state (or a distorted high-energy intermediate) for an enzymatic reaction. Recently, we noted that duplex nucleic acid having a dimethylene sulfone unit replacing a phosphate has a distorted backbone conformation reminiscent of the distortion produced by restriction enzymes and other proteins that bend DNA when they bind. In particular, the twist observed in a duplex built from the dinucleotide analogue r(GSO sub(2)C) is a low 20.8 degree (instead of 34.7 degree ), similar to the twist observed between the central four base pairs in DNA carrying the recognition sequence bound to EcoRV (19.3-23.0 degree ) (Table 1). Thus, a DNA duplex having a dimethylene sulfone substitution joining these base pairs should be "pre-distorted" in its ground state and, therefore, bind more tightly to EcoRV than the cognate DNA substrate itself. For such an oligonucleotide analogue to be an effective inhibitor of the enzyme, the sulfone group must also mimic interactions that the phosphate group has with the enzyme itself. EcoRV makes many contacts to the phosphate groups in its recognition sequence, both directly and via water molecules. As the sulfone S=O bond has a high dipole moment, the sulfone should accept hydrogen bonds, although perhaps not as well as phosphate, where each oxygen bears a charge of ca. -0.5. Solvation of the sulfone group in the GSO sub(2)C duplex is rather similar to solvation of the phosphate group in the GPO sub(2) super(-) sub(C) duplex, providing experimental evidence suggesting that the sulfone group might adequately serve as a neutral phosphate mimic.