Heath H. Herman

Dopamine-B-hydroxylase: Suicide inhibition by the novel olefinic substrate, 1-phenyl-1-aminomethylethene

Dopamine-B-hydroxylase [E.C.1.14.17.1] plays a key role in the biosynthetic interconversion of neurotransmitters. It is now demonstrated for the first time that dopamine-B-hydroxylase also catalyzes the oxygenation of an olefinic substrate, 1-phenyl-1-aminomethylethene, producing 2,3-dihydroxy-2-phenylpropylamine after acid workup. This reaction gives the normal oxygenase stoichiometry of electrons to O2 to product of 2:1:1, and is kinetically comparable to other oxygenase activities of dopamine-B-hydroxylase, with a kcat value of 10 sec-1 and a KM of 8.3 mM. 1-Phenyl-1-aminomethylethene is also a time-dependent, first-order inactivator of dopamine-B-hydroxylase. The inactivation process exhibits the characteristics of mechanism-based, irreversible inactivation, giving a KI value of 13 mM and a kinac of 0.04 min-1. The central role of dopamine-B-hydroxylase in catecholamine metabolism suggests possible pharmacological uses for olefinic inhibitors of this enzyme.

Bioactivation of Cathaedulis alkaloids: Enzymatic ketonization of norpseudoephedrine

Abstract Among the phenylalkylamines which have been shown to have potent central nervous system stimulatory activity are (+)-norpseudoephedrine and its ketone analog, cathinone, and recent work has suggested that cathinone is the more potent agent of the two. We have found that (+)-norpseudoephedrine is oxygenated by dopamine-β-hydroxylase, producing cathinone with the expected stoichiometry of electrons to O 2 to product of 2:1:1 diagnostic of a monooxygenase-catalyzed reaction. A k cat value of 10 sec −1 and K m of 18.8 m M were obtained, indicating that this reaction proceeds readily enough to be of physiological significance. The delayed onset and long duration of action of (+)-norpseudoephedrine is consistent with a requirement for enzyme activation and thus it is possible that dopamine-β-hydroxylase-catalyzed ketonization mediates the potent stimulatory effect of this and related compounds.

Demonstration of the potent antihypertensive activity of phenyl-2-aminoethyl sulfides

In previous work we established that phenyl-2-aminoethyl sulfide (PAES) and derivatives of this basic structure are novel substrate analogs for the adrenergic synthetic enzyme, dopamine beta-monooxygenase (DBM). We examined the in vivo effects of infusions of PAES and ring-hydroxylated (HOPAES) and/or alpha-methylated derivatives (MePAES, HOMePAES) and observed antihypertensive activity in SHR with HOPAES and HOMePAES using an indirect blood pressure measuring protocol. We now wish to report that by employing a direct blood pressure measuring technique we have been able to demonstrate a potent antihypertensive activity of all these derivatives in conscious, unrestrained spontaneously hypertensive rats (SHR) that persisted beyond a 6-h testing period. We found that MePAES, which displayed only a minor antihypertensive activity in the indirect measurements, was the most potent antihypertensive in the direct measuring protocol. In addition, in this report we demonstrate a potent chronic antihypertensive effect for MePAES over a 2-week period in SHR using continuous infusion with implanted osmotic pumps. From a comparison of the effects of the hydroxylated and alpha-methylated derivatives, we conclude that: (a) the locus of the antihypertensive activity is primarily in peripheral adrenergic sites; (b) alpha-methylation of the basic structure imparts an increased affinity for peripheral adrenergic uptake sites that may be responsible for its increased antihypertensive potency; and (c) monoamine oxidase (MAO) catabolism plays a relatively unimportant role in the termination of the activity of these compounds. These results also demonstrate the importance of direct blood pressure measurements in assaying antihypertensive activity of test compounds that possess indirect sympathomimetic activity. The implications of these findings in terms of the mechanism by which these compounds exert their anti-hypertensive activity is discussed.

N-succinimidyl methoxyphenylacetic acid ester, an amine-directed chiral derivatizing reagent suitable for enzymatic scale resolutions

A chiral derivatizing reagent, N-succinimidyl-2-(S)-methoxy-2-phenylacetic acid ester (SMPA), directed toward reaction with primary amine-containing compounds has been synthesized and characterized. This reagent is suitable for HPLC resolution from enzymatic-scale reactions where only microgram quantities of chiral products may be obtainable. SMPA derivatization was shown to be effective in the resolution of the enantiomers of a number of different racemic compounds. SMPA was used to resolve the diastereoisomeric derivatives of a previously unknown enzymatically oxygenated product, allowing determination of the stereochemical course of the enzymatic reaction. SMPA is easily prepared from an inexpensive, commercially available, and enantiomerically pure precursor with the formation of a shelf-stable crystalline product which is utilizable in water-containing solutions. In addition to its usefulness for micro-determinations, SMPA is useful for preparative-scale resolutions of enantiomers since the reagent is cleaved from the diastereoisomeric derivative by acid hydrolysis.

Effects of phenyl-2-aminoethyl sulfide, a novel dopamine-beta-hydroxylase substrate, on the cardiovascular system of the anesthetized dog.

In previous work we have established that phenyl-2-aminoethyl sulfide (PAES) is a novel substrate for dopamine-β-hydroxylase (DBH) which is stereospecifically oxygenated by the enzyme to the corresponding sulfoxide, (S)-phenyl-2-aminoethyl sulfoxide (PAESO). We now report that PAES possesses very little, if any, direct adrenergic agonist activity, but exhibits indirect sympathomimetic activity at relatively high doses (∼4 mg/kg). This assertion, that PAES is a new indirect sympathomimetic, is supported by our finding that pretreatment with cocaine completely abolishes the sympathomimetic activity of PAES. Furthermore, the effects of PAES are diminished with consecutive administration. In addition to its indirect sympathomimetic activity, we have also observed that PAES infusion almost completely blocks the reflex response elicited by hydralazine, a direct vasodilator. In contrast, we have found that PAESO possesses neither direct nor indirect sympathomimetic activity at doses as high as 6 mg/kg. Since PAES should be readily converted in vivo into PAESO, the implications of these findings in terms of potential antihypertensive action of PAES are discussed.