Spontaneous epimerization of (S)-deoxycoformycin and interaction of (R)-deoxycoformycin, (S)-deoxycoformycin, and 8-ketodeoxycoformycin with adenosine deaminase

Publisher Website
Google Scholar
Abstract
(R)-Deoxycoformycin (pentostatin), (S)-deoxycoformycin and 8-ketodeoxycoformycin were compared as inhibitors of calf intestine adenosine deaminase. In contrast to (R)-deoxycoformycin, which was demonstrated as a tight-binding inhibitor with a dissociation constant of 2.5 .times. 10-12 M, (S)-deoxycoformycin and 8-ketodeoxycoformycin are slope-linear competitive inhibitors with respect to adenosine. The kinetic constants are 33 .mu.M for inhibition by (S)-deoxycoformycin, 43 .mu.M for 8-ketodeoxycoformycin and 16 .mu.M for the Km for adenosine. The stereochemistry of carbon 8 of the diazepine ring therefore causes a (1:3 .times. 107)-fold change in the affinity for the enzyme which is specific for the R configuration. This difference is attributed to an induced conformational change which cannot be initiated by the S isomer or the 8-keto analog of (R)-deoxycoformycin. The studies were complicated by the need to remove traces of tight-binding inhibitor(s) from (S)-deoxycoformycin, since as little as 0.001% of the R isomer causes significant inhibition. The R and S isomers of deoxycoformycin are unstable in neutral or mildly acidic aqueous solutions. Isomerization of the secondary hydroxyl at carbon 8 of the diazepine ring is one of the reactions, resulting in S to R and R to S conversions for deoxycoformycins. Opening of the aglycon is also a major reaction. The tight-binding inhibitor generated from (S)-deoxycoformycin was identified as (R)-deoxycoformycin by high-pressure liquid chromatography, spectroscopy, circular dichroism and chemical criteria. The enzyme did not catalyze epimerization of (S)- or (R)-deoxycoformycins. Dialysis of (S)-deoxycoformycin against excess enzyme provided an efficient method for removing (R)-deoxycoformycin or other tight-binding inhibitors. This technique provides a general solution for removing traces of tight-binding inhibitors from larger quantities of weaker inhibitors.