Deuterium isotope effects on in vitro dealkylation of 1,1-dimethylhydrazine and 1,1-dimethylnitrosamine via hepatic enzymes

Boiling 1,1-dimethylnitrosamine or unsymmetrical dimethylnitrosamine several times with 25% NaOD in D2O promoted the complete exchange of the nitrosamine’s alpha protons for deuterium. The deuterated nitrosamine was reduced by LiAlH4, in anhydrous diethyl ether to the corresponding 1,1-dimethylhydrazine-d6. An in-process production of the oxalate salt was necessary due to the air sensitivity of the free base hydrazine. After recrystallization from absolute ethanol and extensive drying, the oxalate salt was evaluated for purity by mixed melting point, high performance liquid chromatography and thin layer chromatography. When protonated hydrazine-oxalate salt (mp=146 °C) was mixed with a sample of the exchanged hydrazine salt there was no melting point depression. Several chromatographic procedures were used to investigate the presence of impurities in the sample. Each process revealed only the presence of the free base hydrazine and oxalic acid. Complete deuterium exchange was characterized by mass spectrometry and 1H-nmr The mass spectum of the 1,1-dimethylhydrazine-d6 had a molecular ion peak at mass 66 relative to the protonated free base hydrazine and the protonated hydrazine-oxalate salt whose molecular ion peak was at mass 60. The subsequent 1H-nmr spectra of both the protonated and deuterated hydrazine-oxalate salts, when dissolved in D2O, exhibited peaks specific for the two protons on the oxalic acid but only the protonated form had two additional absorbance peaks due to the two sets of alpha methyl protons. Using difference absorbance spectroscopy, the interaction of both protonated and deuterated 1,1-dimethylhydrazine with microsomal cytochrome P-450 was examined. As enzyme substrates, both hydrazines exhibited identical difference absorbance spectra when added to suspensions of rat liver microsomes. Also, the resulting difference absorbance spectra were similar to many nitrogenous ligands to cytochrome P-450. With the addition of NADPH, the enzyme suspension containing the hydrazine, formed a time and oxygen dependent microsomal hemoprotein spectral-complex. Both hydrazines formed a spectral-complex that exhibited a Soret absorbance maximum at 438 nm with alpha and beta absorbance bands at 575 and 547 nm. Upon standing the suspensions were depleted of oxygen thus causing a bathochromic shift of the Soret absorbance to give a second complex with a maximum absorbance at 449 nm. Upon subsequent oxygenation of the suspension the 438 nm absorbance was re-established which is consistent for ferric to ferrous transition of the hemoprotein. Both the protonated and deuterated form of the hydrazine formed the 438 nm-complex suggesting that the presence of deuterium had little effect on the mechanism of substrate reduction. Vmax for both substrates was so great however, that kinetic evaluation of the reaction mechanism could not be done. Normally, 1,1-dimethylnitrosamine exhibits a type I difference binding spectra when added to rat liver microsomal suspensions. However, this was not observed for either the protonated or the deuterated form of the nitrosamine. Further changes in the reaction environment failed to yield the type I binding spectra or a 438 nm-complex. Inverse kinetic isotope effects on Vmax and Vmax/Km were observed when 1,1-dimethylhydrazine-d6, instead of 1,1-dimethylhydrazine, was added to o purified liver microsomal flavin-containing monoxygenase, the Zeigler enzyme. Isotope effects on VmaxH/VmaxD, DV were 0.76 without the presence of n-octylamine and 0.82 when n-octylamine was present in the reaction mixture. Isotope effects on Vmax/Km, DV/K were 0.36 without the presence of n-octylamine and 0.55 when n-octylamine was present in the reaction mixture. The inverse isotope effect was observed relative to the protonated substrate when each were added, as substrate, to the purified enzyme and in the presence of NADPH. n-Octylamine, known to increase the rate of Ziegler enzyme reaction, had little effect on the magnitude of the inverse kinetic effect. Possible explanations for this inverse kinetic effect may be due to the deuterium affecting the binding rate of substrate to enzyme. Examination of the acid quenched reaction products revealed the presence of monomethylhydrazine. The formation of the monomethylhydrazine supports the findings by Prough, (11). Prough found the enzyme promoted the formation of a 1,1-dimethyldiazene intermediate, possibly from the dehydration of an N-hydroxymetabolite. Under acid conditions, this tautomerization of the diazene via an azo-intermediate to a methylhydrazone and then on to the monomethylhydrazine is favored over a possible bimolecular reaction involving the diazene which would promote the formation of a corresponding tetramethyltetrazene