A spin-labeled analogue of NADP has been prepared and characterized. Adenosine 2‘,3‘-phosphate 5‘-diphospho-4-(2,2,6,6-tetramethylpiperidinyl-l-oxy) has been prepared by coupling adenosine 2‘,3‘-phosphate 5‘-phosphoromorpholidate with 4-phospho-2,2,6,6-tetramethylpiperidinyl-l-oxy and isolated by ion-exchange chromatography. The cyclic phosphate was cleaved by treatment with dilute acid to give a mixture of the 2‘- and 3‘-phosphate isomers. Incubation with a 3‘-nucleotidase and subsequent ion-exchange chromatography gave the desired 2‘-phosphate isomer, adenosine 2‘-phosphate 5‘-diphospho-4-(2,2,6,6-tetramethylpiperidinyl-l-oxy). The spin-labeled analogue of NADP inhibits dihydrofolate reductase isoenzyme II from Streptococcus faecium var. durans Strain A. The inhibition is competitive with respect to NADPH with Kis = 7.7 ± 1.4 üM. This is consistent with the dissociation constant for the inhibitor complex, KD = 5.1 ± 0.8 üM, as measured by the effect of binding on the EPR signal of the ligand. The electron paramagnetic resonance (EPR) measurements also show that the number of binding sites for the ligand on the reductase is 1.1 ± 0.1. The EPR signal shape at high ratios of the analogue to enzyme is unchanged from the signal in the absence of enzyme, but at low ratios of analogue to enzyme there is a marked broadening of the signal. Analysis of the enzyme-bound signal gave a correlation time of 5 ns, which is somewhat shorter than the rotational correlation of the enzyme [20 ns; Cocco, L., Blakley, R. L., Walker, T. E., London, R. E., & Matwiyoff, N. A. (1978) Biochemistry 17, 4285-4290], The analogue does not appear to inhibit glutamate dehydrogenase, isocitrate dehydrogenase, or glucose-6-phosphate dehydrogenase. © 1979, American Chemical Society. All rights reserved.
