Bernhard Witkop

Role of the arene oxide-oxepin system in the metabolism of aromatic substrates: I. In vitro conversion of benzene oxide to a premercapturic acid and a dihydrodiol

Abstract On incubation with rabbit liver microsomes, benzene oxide is converted enzymatically to trans-1,2-dihydro-l,2-dihydroxybenzene. The enzyme, “epoxide hydrase,” is found in both the microsomal and the soluble fractions and converts a variety of epoxides to 1,2-glycols. Hydration to glycols has been demonstrated for styrene oxide, indene oxide, and cyclohexene oxide. The trans-1,2-dihydro-1,2-dihydroxybenzene is dehydrogenated to catechol by the supernatant of microsomes. In the presence of acid, benzene oxide readily rearranges to phenol. This isomerization is also catalyzed by proteins, simple peptides, and acetamide, but not by N,N-dimethylacetamide. Consequently phenol is a major nonenzymatic product in all enzymatic studies with benzene oxide. An enzyme in the soluble fraction of rat liver catalyzes the addition of glutathione to benzene oxide forming the premercapturic acid, S-(1,2-dihydro-2-hydroxyphenyl)-glutathione.

Batrachotoxin: Chemistry and Pharmacology

Batrachotoxin has been shown to be a pyrrolecarboxylic ester of a novel steroidal base with unique and selective actions on a variety of electrogenic membranes. The effects of batrachotoxin in neuromuscular preparations both pre- and postsynaptically, in nerve axons, in superior cervical ganglion, in heart Purkinje fibers, and in brain slices appear to be due to the selective and irreversible increase in permeability of membranes to sodium ions. The subsequent effects of this increase in Na+ permeability evoked by batrachotoxin—such as membrane depolarization, enhanced spontaneous transmitter release, muscle contracture, and enhanced formation of cyclic AMP in brain slices—may be blocked reversibly by tetrodotoxin.

Nonenzymatic Methods For The Preferential And Selective Cleavage And Modification Of Proteins

Publisher Summary This chapter reviews that there is a growing conviction among organic chemists that the scientific frontier of organic chemistry has moved from small molecules to the large entities that are involved in the process of the formation and preservation of the living cell. Consequently, the skills of the organic chemist, wisely, are applied to the design of the special methods for the analysis, degradation, and synthesis of large molecules. All the reactions utilized for the selective cleavage of the proteins are conceptually not new, and in retrospect, surprisingly simple. In principle, all selective cleavages are variations of a key theme— namely, intramolecular acceleration of displacement or elimination reactions to the point where they occur under extremely mild conditions. The formulation and recognition of this principle, very fruitful for protein chemistry, may stimulate and influence the search for the better methods in neighboring fields. The chapter highlights that the rate of reaction of the oxidant with the reactive products and their utilization for further displacement and elimination reactions are the variables which, when properly manipulated, makes for useful cleavage methods applicable to all three kinds of macromolecules.

Substrates and inhibitors of dopamine-β-oxidase

The enzyme dopamine-β-oxidase not only effects the hydroxylation of dopamine to norepinephrine, but accepts as substrates a wide vareety of phenethylamine derivatives such as epinine, m-tyramine and their branched α-methyl derivatives, m-methoxytyramine, which is converted to normetanephrine, 3,5-dimethoxytyramine, and to some extent even mescaline. The fact that many well-known sympathomemetic drugs are good substrates forthe enzyme raises the pissibility that compounds such as amphetamine, paredrinol, paradrine and α-methyl-m-tyramine owe some of their activity to the corresponding metabolites which are all derivatives of ephedrine. The non-specificity of dopamine-β-oxidase prompted a search for competitive or specific inhibitors. enzylhydrazine, isosteric with phwnethylamine, inhibited the enzyme strongly at concentrations of 10−5M. The therapeutic and clinical implications of this observation are pointed out.

Selective Cleavage and Modification of Peptides and Proteins

Publisher Summary This chapter focuses on the different aspects of selective cleavage and modification of peptides and proteins. The selective chemical cleavages of proteins or peptides currently in use are classified into three main groups based upon the site of cleavage relative to the chemically modified residue: (1) cleavage at the α-carbon atom, (2) cleavage at the amino peptide bond, or (3) cleavage at the carboxyl peptide bond. The theme of participation of neighboring groups for peptide cleavage has stimulated numerous variations, and the search for new oxidants, alkylating reagents, and nucleophiles is still going on. Interest is almost equally divided between the dual aspects of most of these reactions; in addition to cleavage procedures, the need for selective modification of proteins continues. In addition, the knowledge of the complete tertiary structure of important enzymes, such as lysozyme, ribonuclease, α-chymotrypsin, and carboxypeptidase, has permitted a sober evaluation of the limitations of the so-called “group-specific reagents” in protein chemistry and has shown the extent of difficulty in finding an easy correlation between chemical reactivity and tertiary structure.

The combined use of α-methyltyrosine and threo-dihydroxyphenylserine—selective reduction of dopamine levels in the central nervous system

Abstract Dihydroxyphenylserine- α - 14 C (DOPS- α - 14 ) is taken up into mouse brain and decarboxylated to form norepinephrine- 14 C. The combined use of nontracer doses of this amino acid ( threo- or erythro-DOPS ) and the tyrosine hydroxylase inhibitor, α-methyltyrosine ethyl ester, causes a 50 per cent depletion of dopamine in the central nervous system in mice while leaving norepinephrine levels unchanged. Without threo -DOPS, α-methyltyrosine depletes both norepinephrine and dopamine levels to less than one-half their normal values. dl - Threo -DOPS rapidly replenishes norepinephrine levels in reserpine-treated mice, but does not cause the awakening effect shown by l -DOPA under the same conditions. It is suggested that the reversal of the reserpine syndrome by l -DOPA is due to formation of dopamine rather than norepinephrine in the central nervous system.

Migration of deuterium during hydroxylation of aromatic substrates by liver microsomes: I. Influence of ring substituents

Abstract The hydroxylation of a variety of specifically deuterated aromatic substrates by liver microsomes has been investigated. Labeled substrates which cannot readily ionize by loss of a proton from the ring substituent display deuterium retentions varying from 40 to 64% upon hydroxylation at the ring position bearing the isotopic label. These substrates include compounds with either electron-donating or electron attracting substituents (anisole-4- 2 H, diphenyl ether-4- 2 H, biphenyl-4- 2 H, N -methyl- N -(phenyl-4- 2 H)-benzenesulfonamide, toluene-4- 2 H, fluorobenzene-4- 2 H, chlorobromobenzene-4- 2 H, benzonitrile-4- 2 H, benzamide-4- 2 H, and nitrobenzene-4- 2 H). Substrates that can, however, ionize by loss of a proton from the ring substituent exhibit lower retentions ranging from 0 to 30% at pH 8. This class of compounds includes phenols such as salicylic-5- 2 H acid, aniline-4- 2 H, N -(phenyl-4- 2 H)-benzenesulfonamide, and various N -acylanilines-4- 2 H. As the acidity of the amide hydrogen increases in the acylanilines, migration and retention of deuterium decreases. A mechanism is presented which accommodates the retention values observed for both classes of substrates. The degree of retention for acetanilide-4- 2 H is strongly dependent upon the pH of the incubating medium ranging from 21 (pH 10) to 44% (pH 6). N -(Phenyl-4- 2 H)-benzamide shows a similar pH dependence whereas nonionizable substrates (biphenyl-4- 2 H or anisole-4- 2 H) do not display this dependence. The relative reactivity of the substrates studied toward the microsomal hydroxylating system correlates well with their reactivity toward chemical reagents which cause electrophilic substitution: i.e., reactive or electron-rich rings act as the best substrates. In chemical terms, the microsomal hydroxylating system acts as though it is a weak, selective electrophile.

The venom of the Colombian arrow poison frogPhyllobates bicolor

Ein hochaktives Gift mit einer LD50 von mindestens 2.7 ± 0.2 µg/kg Maus wurde aus der Haut des columbischen PfeilgiftfroschesPhyllobates bicolor isoliert. Nach 60 facher Anreicherung erwies sich das Produkt in der Dünnschichtchromatographie als einheitlich. Es ist löslich in organischen Lösungsmitteln wie Chloroform und Methylenchlorid und lässt sich aus solchen Lösungen mit wässriger Säure extrahieren. Aus dem Verteilungsverhalten der Aktivität bei verschiedenen pH-Werten wurden pK-Werte von 7.1 und 8.0 ermittelt, die auf die Anwesenheit eines basischen Strukturelementes hinweisen. Das UV-Spektrum zeigt Endabsorption mit Schultern bei 220, 230 und 260 mµ. Eine ausgeprägte Absorptionsbande im IR-Spektrum (CHCl3) bei 1690 cm−1 deutet auf eine Amidcarbonylgruppe hin. Das Gift bewirkt im Nerv-Muskelpräparat zunächst eine irreversible Blockierung der Nervenendplatten; später folgen myotrope Effekte (Kontraktur).In vivo wird neben der Atemlähmung eine starke, wahrscheinlich zentral ausgelöste Krampfwirkung neben anderen Effekten beobachter, die das Kokoigift deutlich vom Curare abhebt. Das Kokoigift ist das stärkste bis jetzt bekannte Gift animalischen Ursprungs.

Synthetic polypeptides as substrates and inhibitors of collagen proline hydroxylase

Abstract The synthetic polypeptide ([3,4- 3 H-Pro]-Gly-Pro) n can be hydroxylated by collagen proline hydroxylase. The affinity of the enzyme for the substrate increases with increasing molecular weight of substrate over the range 1300–8000. The data indicate that maximal affinity should be observed with a polypeptide of molecular weight considerably greater than 8000. Poly-L-proline II and (Pro-Gly-Pro) n appear to be com petitive inhibitors of the hydroxylation reaction. The series of oligopeptides, (Glv Pro-Ala) n , (Gly-Pro-MePro) n , (Pro-Gly-Pro) n , and (Pro-Gly-FPro) n Pro, were tested for inhibition of hydroxylation over the range n = 1 to n = 4. Inhibition is first observed at n = 2 and for these short oligopeptides is maximal between n = 3 and n = 4. Secondary structure appears to be less important than primary structure in determining whether a peptide will be an inhibitor of the hydroxylase. It is suggested that in a polypeptide chain a specific sequence of 6–9 amino acids is required to identify an appropriate prolyl residue for hydroxylation. Among a series of proline derivatives tested, only 4-fluoro, 3- and 4-methylproline inhibited the hydroxylase.

Hydroxylation of alkyl and halogen substituted anilines and acetanilides by microsomal hydroxylases

Abstract The hydroxylation of a variety of halo and alkyl substituted anilines and acetanilides with hepatic microsomal preparations was studied. 4 Chloro- and 4-fluoro-acetanilides and anilines were hydroxylated to the corresponding 4-hydroxy derivatives with loss of halogen. The 4-chloro derivatives were extremely poor substrates for the aryl hydroxylases. 3-Methyl-, 3-chloro-, and 3-fluoro-acetanilides were metabolized to the corresponding 4-hydroxy-3-substituted acetanilide. 4-Methylaniline, 4-methyl-acetanilide, and 4-ethylacetanilide were not substrates for aryl hydroxylation, but instead underwent side-chain oxidation to form, respectively, 4-hydroxymethylaniline, 4-hydroxymethylacetanilide, and 4-(1′-hydroxyethyl)acetanilide. The hydroxymethyl compounds were further oxidized to aldehydes.